Methods for treating heart failure using glucagon receptor antagonistic antibodies

ABSTRACT

The present disclosure relates to methods for treating or preventing heart failure using a glucagon receptor blocking agent. In various embodiments, the present disclosure relates to methods for treating or preventing heart failure (e.g., post-myocardial infarction heart failure), diabetic cardiomyopathy heart failure), and lateral ventricular (LV) remodeling, using antigen binding and antagonizing proteins, e.g., fully human antibodies, that specifically bind to and antagonize the function of the human glucagon receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 15/757,352, filed Mar. 3, 2018, which is a U.S. National StageApplication pursuant to 35 U.S.C. § 371 of PCT/US2016/050183, filed Sep.2, 2016, which claims priority to provisional application U.S. Ser. No.62/214,449, filed Sep. 4, 2015, and U.S. Ser. No. 62/259,061, filed Nov.24, 2015, each hereby incorporated by reference in their entirety.

BACKGROUND

Heart failure (HF) is a global problem with an estimated prevalence of38 million patients worldwide, including 6 million in the United Statesand more than 550,000 new patients diagnosed with the condition in theUS each year. HF is the most common diagnosis in patients aged 65 yearsor older admitted to hospital and it is one of the most significantcauses of morbidity and mortality in developed countries.

Heart failure is a clinical syndrome characterized by the failure of theheart to pump sufficient blood to meet the body's systemic demands. Theheart contracts and relaxes with each heartbeat—these phases arereferred to as systole (the contraction phase) and diastole (therelaxation phase). Systolic heart failure (SHF) is characterized by lowejection fraction. In patients with diastolic heart failure (DHF),contraction may be normal but relaxation of the heart may be impaired.This impairment is generally caused by a stiffening of the ventricles.Such impairment is referred to as diastolic dysfunction and if severeenough to cause pulmonary congestion (increased pressure and fluid inthe blood vessels of the lungs), diastolic heart failure. DHF patientsdiffer from those patients with SHF, in that DHF patients may have a“normal” ejection fraction. However, because the ventricle doesn't relaxnormally, the pressure within the ventricle increases and the bloodfilling the ventricle exceeds what is “normal”. People with certaintypes of cardiomyopathy may also have diastolic dysfunction.

Left ventricular hypertrophy (LVH) refers to a thickening of the leftventricle as a result of increased left ventricular load. LVH can be asignificant marker for cardiovascular disorders and most commoncomplications include arrhythmias, heart failure, ischemic heartdisease, and sudden death. Although LVH increases naturally with age, itis more common in people who have high blood pressure or have otherheart problems. Because LVH usually develops in response tohypertension, current treatment and prevention mainly includes managinghypertension. Typical diagnosis involves the use of echocardiograms(ECHO) and electrocardiograms (ECG).

Myocardial infarction (MI) is a leading cause for heart failure. Themechanism of an MI often involves the rupture of an atheroscleroticplaque leading to complete blockage of a coronary artery which resultedin the death or damage of heart muscle cells because the heart musclecells do not receive enough oxygen. Diabetes mellitus (type 1 or 2),high blood pressure, dyslipidemia/high levels of blood cholesterol,particularly high low-density lipoprotein, low high-density lipoprotein,high triglycerides, and obesity have all been linked to myocardialinfarction (Jay N. Cohn, et al., Journal of the American College ofCardiology, 35(3), 569-82, 2000). Long-term outcome after MI can belargely be defined in terms of its impact on the size and shape of theleft ventricle (i.e., LV remodeling). Three major mechanisms contributeto LV remodeling: i) early infarct expansion, ii) subsequent infarctextension into adjacent non infarcted myocardium, and iii) latehypertrophy in the remote LV (Id). As the heart remodels, it not onlygets bigger, but the cardiac walls get thinner and the pumping capacityof the heart declines. Cardiac remodeling is generally accepted as adeterminant of survival after recovery from MI. Although the importanceof remodeling as a pathogenic mechanism is incompletely understood,cardiac remodeling is thought to be an important aspect of diseaseprogression in HF, regardless of cause (Id). Left ventricular remodelingis the process by which ventricular size, shape, and function areregulated by mechanical, neuro-hormonal, and genetic factors. Remodelingmay be physiological and adaptive during normal growth or pathologicaldue to MI, cardiomyopathy, hypertension, or valvular heart disease(French and Kramer, Drug Discov. Today Dis Mech., 4(3): 185-196, 2007).

It was recently reported that glucagon receptor signaling incardiomyocytes modulates outcomes in non-diabetic mice with experimentalmyocardial infarction (Ali, et al., Molecular Metabolism, 4:132-143,2015). Specifically, exogenous glucagon administration directly impairedrecovery of ventricular pressure in ischemic mouse hearts ex vivo, andincreased mortality from myocardial infarction after LAD coronary arteryligation in mice in a p38 MAPK-dependent manner. In contrast,cardiomyocyte specific reduction of glucagon action in adultGCGR^(CM−/−) mice (mice having GCGR inactivated) significantly improvedsurvival, and reduced hypertrophy and infarct size following myocardialinfarction (Id.) The authors conclude that the cardiovascularconsequences of manipulating glucagon action remain poorly understoodand emphasize the need to acquire a further understanding of suchconsequences (Id.)

Current drug treatments available for management of heart failure, andother cardiovascular disorders, include vasodilators to reduce the bloodpressure and ease the workload of the heart, diuretics to reduce fluidoverload, inhibitors and blocking agents of the body's neuro-hormonalresponses (e.g., angiotensin-converting enzyme (ACE) inhibitors andbeta-adrenergic blocking agents), and other medicaments. Suchmedications, while effective for a short time, often cannot be used forextended periods because of side effects. Various surgical proceduressuch as heart transplantation have also been proposed for patients whosuffer from severe, refractory heart failure. Alternatively, animplantable medical device such as ventricular assist devices (VADs) maybe implanted in the chest to increase the pumping action of the heart,or an intra-aortic balloon pump (IABP) may be used for maintaining heartfunction for short periods of time, but typically no longer than onemonth. While each of these approaches have proven to be at least partlybeneficial to patients, they each have shortcomings which limit theiroverall effectiveness. For example, drug therapies often involveunwanted side effects and complex therapy regimens which contribute topoor patient compliance. And both drug therapy and surgical approachesare very costly, adding to the health care costs associated with heartfailure. Despite the ongoing research and development of treatments forheart failure, there is still a tremendous need for improved andalternative treatments.

SUMMARY OF THE INVENTION

The present disclosure is based in part on the inventors' unique insightthat isolated antigen binding and antagonizing proteins thatspecifically bind to the human glucagon receptor may provide forimproved, effective therapies for treatment and prevention of heartfailure after myocardial infarction, and prevention of heart failureassociated with diabetes and hyperglycemia. The present inventorproposes that the beneficial therapeutic effects provided by blockingthe glucagon receptor in myocardial infarction-induced heart failuresubjects (or all other heart failure subjects) relate to the preventionor attenuation of left ventricular (LV) remodeling which may include:increasing fractional shortening (FS), decreasing LV dilation (LVESD);preserving LV wall thickness; increasing LV developed pressure andventricular contractility; decreasing the ratio of heart weight to bodyweight (infarct size); reducing the fibrosis process; reducing levels ofincompletely oxidized fatty acid metabolites in the heart under ischemicconditions; and reducing MI-induced mortality rate.

Thus, in one aspect, the present disclosure comprises a method fortreating or preventing heart failure and associated conditions in asubject, comprising administering to a subject diagnosed with heartfailure, or a subject at risk of contracting heart failure, atherapeutically effective amount of an isolated antagonistic antigenbinding protein that specifically binds to the human glucagon receptor.In various embodiments, the present disclosure comprises a method fortreating or preventing heart failure after myocardial infarction. Invarious embodiments, the present disclosure comprises a method fortreating or preventing heart failure associated with diabetes mellitus.In various embodiments, the present disclosure comprises a method fortreating or preventing heart failure associated with diabeticcardiomyopathy.

In various embodiments, the isolated antagonistic antigen bindingprotein comprises an antibody selected from a fully human antibody, ahumanized antibody, a chimeric antibody, a monoclonal antibody, apolyclonal antibody, a recombinant antibody, an antigen-binding antibodyfragment, a Fab, a Fab′, a Fab₂, a Fab′₂, a IgG, a IgM, a IgA, a IgE, ascFv, a dsFv, a dAb, a nanobody, a unibody, or a diabody. In variousembodiments, the antibody is a fully human monoclonal antibody. Invarious embodiments, the isolated antibody or antigen-binding antibodyfragment specifically binds to a human glucagon receptor with adissociation constant (K_(D)) of at least about 1×10⁻⁷ M, at least about1×10⁻⁸ M, at least about 1×10⁻⁹ M, at least about 1×10⁻¹⁰ M, at leastabout 1×10⁻¹¹ M, or at least about 1×10⁻¹² M.

In various embodiments, the isolated antagonistic antigen bindingprotein comprises an antibody which comprises the amino acid sequenceencoding the heavy chain variable region of SEQ ID NO: 2 and the aminoacid sequence encoding the light chain variable region of SEQ ID NO: 3.In various embodiments, the isolated antagonistic antigen bindingprotein comprises an antibody which comprises the amino acid sequenceencoding the heavy chain variable region of SEQ ID NO: 4 and the aminoacid sequence encoding the light chain variable region of SEQ ID NO: 5.In various embodiments, the isolated antagonistic antigen bindingprotein comprises an antibody which comprises the amino acid sequenceencoding the heavy chain variable region of SEQ ID NO: 6 and the aminoacid sequence encoding the light chain variable region of SEQ ID NO: 7.In various embodiments, the isolated antagonistic antigen bindingprotein comprises an antibody which comprises the amino acid sequenceencoding the heavy chain of SEQ ID NO: 8 and the amino acid sequenceencoding the light chain of SEQ ID NO: 9. In various embodiments, theisolated antagonistic antigen binding protein comprises an antibodywhich comprises the amino acid sequence encoding the heavy chainvariable region selected from the group consisting of SEQ ID NOs: 10-28,and the amino acid sequence encoding the light chain variable regionselected from the group consisting of SEQ ID NOs: 29-47. In variousembodiments, the isolated antagonistic antigen binding protein comprisesan antibody which comprises the amino acid sequence encoding the heavychain of SEQ ID NO: 51 and the amino acid sequence encoding the lightchain of SEQ ID NO: 52.

In various embodiments, the present disclosure comprises a method fortreating or preventing heart failure and associated conditions in asubject, comprising administering to a subject diagnosed with heartfailure, or a subject at risk of developing heart failure, atherapeutically effective amount of an isolated antagonistic antigenbinding protein that specifically binds to the human glucagon receptor;and (b) a second agent composition. In various embodiments, the secondagent composition is selected from a group consisting of:angiotensin-converting enzyme (ACE) inhibitors, β-adrenergic blockingagents, angiotension II receptor blockers (ARBs), diuretics, anddigitalis. In various embodiments, the isolated antagonistic antigenbinding protein comprises an antibody which comprises the amino acidsequence encoding the heavy chain of SEQ ID NO: 51 and the amino acidsequence encoding the light chain of SEQ ID NO: 52.

In various embodiments, the present disclosure comprises a method fortreating or preventing diabetes induced cardiomyopathy in a subject,comprising administering to a subject diagnosed with diabetes, or asubject at risk of developing diabetes, a therapeutically effectiveamount of an isolated antagonistic antigen binding protein thatspecifically binds to the human glucagon receptor; and (b) a secondagent composition. In various embodiments, the second agent compositionis selected from a group consisting of: angiotensin-converting enzyme(ACE) inhibitors, β-adrenergic blocking agents, angiotension II receptorblockers (ARBs), diuretics, and digitalis. In various embodiments, theisolated antagonistic antigen binding protein comprises an antibodywhich comprises the amino acid sequence encoding the heavy chain of SEQID NO: 51 and the amino acid sequence encoding the light chain of SEQ IDNO: 52.

In various embodiments, the isolated antagonistic antigen bindingprotein that specifically binds the human glucagon receptor will beadmixed with a pharmaceutically acceptable carrier to form apharmaceutical composition that can be systemically administered to thesubject via intravenous injection, intramuscular injection, subcutaneousinjection, intraperitoneal injection, transdermal injection,intra-arterial injection, intrasternal injection, intrathecal injection,intraventricular injection, intraurethral injection, intracranialinjection, intrasynovial injection or via infusions.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. FIG. 1 is a line plot depicting the in vivoeffects on mortality for the control (CON) group (n=18), REMD2.59 (mAb)treated group (n=18), and glucagon (GLC) treated group (n=28) throughouta 28 day study wherein myocardial infarction (MI) was induced in 54C57BL6 male mice. Survival proportion is plotted vs. time (days).

FIG. 2 is a line plot depicting the in vivo effects on left ventricularfractional shortening (FS) for the control (CON) group (n=10), REMD2.59(mAb) treated group (n=11), and glucagon (GLC) treated group (n=6)throughout a 28 day study wherein myocardial infarction (MI) was inducedin 54 C57BL6 male mice. % FS is plotted vs. time (weeks). #: P<0.05 Convs GLC, **: P<0.01 mAb vs GLC, ***: P<0.001 mAb vs CON or GLC.

FIG. 3 is a line plot depicting the in vivo effects on left ventricularend systolic diameter (LVESD) for the control (CON) group (n=10),REMD2.59 (mAb) treated group (n=11), and glucagon (GLC) treated group(n=6) throughout a 28 day study wherein myocardial infarction (MI) wasinduced in 54 C57BL6 male mice. LVESD (mm) is plotted vs. time (weeks).#: p<0.05 vs GLC, *: p<0.05 vs CON, **: p<0.01 vs GLC.

FIG. 4 is a line plot depicting the in vivo effects on left ventricularend diastolic dimension (LVEDD) for the control (CON) group (n=10),REMD2.59 (mAb) treated group (n=11), and glucagon (GLC) treated group(n=6) throughout a 28 day study wherein myocardial infarction (MI) wasinduced in 54 C57BL6 male mice. LVEDD (mm) is plotted vs. time (weeks).

FIG. 5 is a line plot depicting the in vivo effects on ischemic leftventricular wall thickness (IVSd) for the control (CON) group (n=10),REMD2.59 (mAb) treated group (n=11), and glucagon (GLC) treated group(n=6) throughout a 28 day study wherein myocardial infarction (MI) wasinduced in 54 C57BL6 male mice. IVEDD (mm) is plotted vs. time (weeks).**: P<0.01 vs CON or GLC, ***: P<0.001 vs CON or GLC, #: P<0.05 Con vsGLC.

FIG. 6 is a bar graph depicting the in vivo effects on left ventriculardiastole pressure (LVDP) for the control (CON) group, REMD2.59 (mAb)treated group, and glucagon (GLC) treated group in a study whereinmyocardial infarction (MI) was induced in 54 C57BL6 male mice. LVDP (mmHg) at 28 days is depicted.

FIG. 7 contains bar graphs depicting the in vivo effects on cardiaccontractility (+dp/dtm-mm Hg/s)(left panel) and relaxation (−dp/dtm-mmHg/s)(right panel) for the control (CON) group, REMD2.59 (mAb) treatedgroup, and glucagon (GLC) treated group in a study wherein myocardialinfarction (MI) was induced in 54 C57BL6 male mice. Cardiaccontractility (+dp/dtm-mm Hg/s)(left panel) and relaxation (−dp/dtm-mmHg/s)(right panel) at 28 days is depicted.

FIG. 8 are echo readouts and corresponding pictures depicting the invivo effects on cardiac remodeling for the control (CON) group, REMD2.59(mAb) treated group, and glucagon (GLC) treated group at day 28 of astudy wherein myocardial infarction (MI) was induced in 54 C57BL6 malemice.

FIG. 9 is a bar graph depicting the in vivo effects on cardiacremodeling for the control (CON) group, REMD2.59 (mAb) treated group,and glucagon (GLC) treated group at day 28 of a study wherein myocardialinfarction (MI) was induced in 54 C57BL6 male mice. The ratio of Heartweight (HW)(mg) vs. Body weight (BW)(g) at 28 days is depicted.

FIG. 10 is a bar graph depicting the in vivo effects on blood glucoselevels for the control (CON) group and REMD2.59 (mAb) treated group atday 14 of a study wherein myocardial infarction (MI) was induced in 54C57BL6 male mice. Blood glucose levels (mg/dl) (fasted for 4 hours) at14 days is depicted. *: P<0.01.

FIG. 11 is a bar graph depicting in vivo effects on apoptosis in theinfarct area for the control (CON) group, REMD2.59 (mAb) treated group,and glucagon (GLC) treated group at day 28 of a study wherein myocardialinfarction (MI) was induced in 54 C57BL6 male mice. 8 fields in eachmouse were counted. % TUNEL positive cells at 28 days is depicted.

FIG. 12 depicts histological H&E staining of various sections depictingin vivo effects on fibrosis in the non-infarct area for the control(CON) group, REMD2.59 (mAb) treated group, and glucagon (GLC) treatedgroup at day 28 of a study wherein myocardial infarction (MI) wasinduced in 54 C57BL6 male mice.

FIG. 13 is a bar graph depicting in vivo effects on fibrosis in thenon-infarct area for the control (CON) group, REMD2.59 (mAb) treatedgroup, and glucagon (GLC) treated group at day 28 of a study whereinmyocardial infarction (MI) was induced in 54 C57BL6 male mice. 8 fieldsin each mouse were counted. % fibrotic area at 28 days is depicted.

FIG. 14 depicts histological H&E staining of various sections depictingin vivo effects on myocyte hypertrophy in the non-infarct area for thecontrol (CON) group, REMD2.59 (mAb) treated group, and glucagon (GLC)treated group at day 28 of a study wherein myocardial infarction (MI)was induced in 54 C57BL6 male mice. Magnification 40×10.

FIG. 15 is a bar graph depicting in vivo effects on myocyte hypertrophyin the non-infarct area for the control (CON) group, REMD2.59 (mAb)treated group, and glucagon (GLC) treated group at day 28 of a studywherein myocardial infarction (MI) was induced in 54 C57BL6 male mice. 8fields in each mouse were counted. Myocyte CSA (μm²) at 28 days isdepicted.

FIG. 16 is a bar graph and corresponding pictures depicting the in vivoeffects on infarct size for the control (CON) group, REMD2.59 (mAb)treated group, and glucagon (GLC) at day 28 of a study whereinmyocardial infarction (MI) was induced in 54 C57BL6 male mice. Ratio (%)of length of infarcted wall to LV wall at 28 days is depicted.

FIG. 17 is a bar graph and corresponding pictures depicting the in vivoeffects on infarct area for the control (CON) group, REMD2.59 (mAb)treated group, and glucagon (GLC) at day 28 of a study whereinmyocardial infarction (MI) was induced in 54 C57BL6 male mice. Ratio (%)of length of infarcted area to total ventricular area at 28 days isdepicted.

FIG. 18 is a line plot depicting the blood glucose levels (mg/dl) forthe db/db (PBS), db/db (mAb), db/+ (PBS), and db/+ (mAb) groupsthroughout the 18 week study. Each mouse was fasting 6 hours, andrepeated measurement ANOVA was used for statistical analysis. ***:p<0.0001 compared db/db (mAb) vs db/db (PBS); ###: p<0.0001 compare db/+(PBS) or db/+ (mAb) vs db/db (PBS).

FIG. 19 is a line plot depicting the results of an oral glucosetolerance test (OGTT) for the db/db (PBS), db/db (mAb), db/+ (PBS), anddb/+ (mAb) groups at week 18 week after treatment with mAb or PBS. Eachmouse received 2.0 g/kg of glucose with gavage. Following oraladministration of 2 g/kg glucose, the blood glucose levels were measuredat different time points (30, 60, 120 min) by using Accu-Chek PerformaSystem. Serum glucose levels (mg/dl) is plotted vs. time (minutes) usingrepeated measurement ANOVA for statistical analysis. ***: p<0.0001compared db/db (mAb) vs db/db (PBS); ###: p<0.0001 compare db/+ (PBS) ordb/+ (mAb) vs db/db (PBS).

FIG. 20 is a line plot depicting the in vivo effects on left ventricularend systolic diameter (LVESD) for the db/db (PBS), db/db (mAb), db/+(PBS), and db/+ (mAb) groups throughout the 18 week study. LVESD (mm) isplotted vs. time (weeks) using repeated measurement ANOVA forstatistical analysis. ***: p<0.0001 compared db/db (mAb) vs db/db (PBS);###: p<0.0001 compare db/+ (PBS) or db/+ (mAb) vs db/db (PBS).

FIG. 21 is a line plot depicting the in vivo effects on left ventricularend diastolic dimension (LVEDD) for the db/db (PBS), db/db (mAb), db/+(PBS), and db/+ (mAb) groups throughout the 18 week study. LVEDD (mm) isplotted vs. time (weeks). P values, ##: p<0.01, compared db/+ (mAb) vs.db/db (PBS); N.S: there is no significant differences compared to db/db(PBS) group based on repeated measurement ANOVA, Bonferroni/Dunn method.

FIG. 22 is a line plot depicting the in vivo effects on left ventricularfractional shortening (FS) for the db/db (PBS), db/db (mAb), db/+ (PBS),and db/+ (mAb) groups throughout the 18 week study using repeatedmeasurement ANOVA to compare whole time points between groups. ***:p<0.0001, compared db/db (mAb) vs. db/db (PBS), ###: p<0.0001, compareddb/+ (PBS) or db/+ (mAB) vs. db/db (PBS).

FIG. 23 is a line plot depicting the in vivo effects on interventricularseptal end diastole (IVSD) for the db/db (PBS), db/db (mAb), db/+ (PBS),and db/+ (mAb) groups throughout the 18 week study using repeatedmeasurement ANOVA for statistical analysis. P values, ***: p<0.0001compared db/db (mAb) vs db/db (PBS); ###: p<0.0001 compare db/+ (PBS) ordb/+ (mAb) vs db/db (PBS).

FIG. 24 is a line plot depicting the in vivo effects on the ratio ofEarly (E) to late (A) ventricular filling velocities (E/A) for the db/db(PBS), db/db (mAb), db/+ (PBS), and db/+(mAb) groups throughout the 18week study using repeated measurement ANOVA for statistical analysis.***: p<0.0001 compared db/db (mAb) vs db/db (PBS); ###: p<0.0001 comparedb/+(PBS) or db/+ (mAb) vs db/db (PBS).

FIG. 25 is a line plot depicting the in vivo effects on Cardiac Output(CO, ml/min) for the db/db (PBS), db/db (mAb), db/+ (PBS), and db/+(mAb) groups throughout the 18 week study using repeated measurementANOVA for statistical analysis. ***: p<0.0001 compared db/db (mAb) vsdb/db (PBS); ###: p<0.0001 compare db/+ (PBS) or db/+ (mAb) vs db/db(PBS).

FIG. 26 is a line plot depicting the in vivo effects on IsovolumicRelaxation Time (IVRT, ms) for the db/db (PBS), db/db (mAb), db/+ (PBS),and db/+ (mAb) groups throughout the 18 week study using repeatedmeasurement ANOVA for statistical analysis. ***: p<0.0001 compared db/db(mAb) vs db/db (PBS); ###: p<0.0001 compare db/+ (PBS) or db/+ (mAb) vsdb/db (PBS).

FIG. 27 is a line plot depicting the in vivo effects on Heart Beat Rate(HR, beat/min) for the db/db (PBS), db/db (mAb), db/+ (PBS), and db/+(mAb) groups throughout the 18 week study using repeated measurementANOVA methods. P values: N.S: there is no statistical significantdifferences compare db/db (mAb) vs. db/db (PBS) group; ###: p<0.0001,compared db/+ (PBS) or db/+ (mAb) vs. db/db (PBS) group; **: compareddb/+ (PBS) or db/+ (mAb) vs. db/db (mAb) group.

FIG. 28 is a bar graph depicting in vivo effects on Left ventriculardiastole pressure (LVDP, mmHg) measured by Mikro-tip catheter for thedb/db (PBS), db/db (mAb), db/+ (PBS), and db/+ (mAb) groups at week 18week. ***: p<0.0001 compared to db/db(PBS); ###: p<0.001 compared todb/db (PBS).

FIG. 29 is a bar graph depicting in vivo effects on +dp/dtm and −dp/dtmvalues (measured by Mikro-tip catheter) for the db/db (PBS), db/db(mAb), db/+ (PBS), and db/+ (mAb) groups at week 18 using repeatedmeasurement ANOVA methods. ***: p<0.0001. **: p<0.01 compared todb/db(PBS); #: p<0.05: ###: p<0.001 compared to db/db (PBS).

FIG. 30 is a bar graph depicting in vivo effects on serum active GLP-1and GHbAc1 levels for the db/db (PBS), db/db (mAb), db/+ (PBS), and db/+(mAb) groups at week 18 using repeated measurement ANOVA methods. Pvalues, * p<0.05; ***: p<0.001.

FIG. 31 is a bar graph depicting in vivo effects on heart weight andbody weight for the db/db (PBS), db/db (mAb), db/+ (PBS), and db/+ (mAb)groups at week 18 using repeated measurement ANOVA methods. P values:**: p<0.01, N.S: no significant difference.

FIG. 32 is a bar graph depicting in vivo effects on blood insulin levels(ng/ml) post treatment at week 18 for the db/db (PBS), db/db (mAb), db/+(PBS), and db/+ (mAb) using repeated measurement ANOVA methods.

MODE(S) FOR CARRYING OUT THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those commonly used and well known in the art. The methodsand techniques of the present disclosure are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Green and Sambrook et al. Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2012) incorporated herein by reference, and Ausubelet al., Current Protocols in Molecular Biology, Greene PublishingAssociates (1992), and Harlow and Lane Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990),incorporated herein by reference. Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications, ascommonly accomplished in the art or as described herein. Thenomenclature used in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are thosecommonly used and well known in the art. Standard techniques are usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of subjects.

Definitions

The terms “peptide” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric. In certain embodiments, “peptides”, “polypeptides”, and“proteins” are chains of amino acids whose alpha carbons are linkedthrough peptide bonds. The terminal amino acid at one end of the chain(amino terminal) therefore has a free amino group, while the terminalamino acid at the other end of the chain (carboxy terminal) has a freecarboxyl group. As used herein, the term “amino terminus” (abbreviatedN-terminus) refers to the free α-amino group on an amino acid at theamino terminal of a peptide or to the α-amino group (imino group whenparticipating in a peptide bond) of an amino acid at any other locationwithin the peptide. Similarly, the term “carboxy terminus” refers to thefree carboxyl group on the carboxy terminus of a peptide or the carboxylgroup of an amino acid at any other location within the peptide.Peptides also include essentially any polyamino acid including, but notlimited to, peptide mimetics such as amino acids joined by an ether asopposed to an amide bond.

The term “therapeutic protein” refers to proteins, polypeptides,antibodies, peptides or fragments or variants thereof, having one ormore therapeutic and/or biological activities. Therapeutic proteinsencompassed by the invention include but are not limited to, proteins,polypeptides, peptides, antibodies, and biologics. (The terms peptides,proteins, and polypeptides are used interchangeably herein.) It isspecifically contemplated that the term “Therapeutic protein”encompasses antibodies and fragments and variants thereof.

Polynucleotide and polypeptide sequences are indicated using standardone- or three-letter abbreviations. Unless otherwise indicated,polypeptide sequences have their amino termini at the left and theircarboxy termini at the right, and single-stranded nucleic acidsequences, and the top strand of double-stranded nucleic acid sequences,have their 5′ termini at the left and their 3′ termini at the right. Aparticular section of a polypeptide can be designated by amino acidresidue number such as amino acids 80 to 119, or by the actual residueat that site such as Ser80 to Ser119. A particular polypeptide orpolynucleotide sequence also can be described by explaining how itdiffers from a reference sequence.

Polypeptides of the disclosure include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties. For example, single or multiple amino acidsubstitutions (e.g., conservative amino acid substitutions) may be madein the naturally occurring sequence (e.g., in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts). A“conservative amino acid substitution” refers to the substitution in apolypeptide of an amino acid with a functionally similar amino acid. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another:

Alanine (A), Serine (S), and Threonine (T)

Aspartic acid (D) and Glutamic acid (E)

Asparagine (N) and Glutamine (Q)

Arginine (R) and Lysine (K)

Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

A “non-conservative amino acid substitution” refers to the substitutionof a member of one of these classes for a member from another class. Inmaking such changes, according to certain embodiments, the hydropathicindex of amino acids may be considered. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in certain embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In certain embodiments,those that are within ±1 are included, and in certain embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1);glutamate (+3.0.+−0.1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5) and tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, in certain embodiments, the substitutionof amino acids whose hydrophilicity values are within +2 is included, incertain embodiments, those that are within +1 are included, and incertain embodiments, those within +0.5 are included. Exemplary aminoacid substitutions are set forth in Table 1.

TABLE 1 Original Exemplary Preferred Residues SubstitutionsSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln AspGlu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn,Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine LeuNorleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric ArgAcid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu ProAla Gly Ser Thr, Ala, Cys Thr Thr Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. In certainembodiments, one skilled in the art may identify suitable areas of themolecule that may be changed without destroying activity by targetingregions not believed to be important for activity. In other embodiments,the skilled artisan can identify residues and portions of the moleculesthat are conserved among similar polypeptides. In further embodiments,even areas that may be important for biological activity or forstructure may be subject to conservative amino acid substitutionswithout destroying the biological activity or without adverselyaffecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, the skilledartisan can predict the importance of amino acid residues in apolypeptide that correspond to amino acid residues important foractivity or structure in similar polypeptides. One skilled in the artmay opt for chemically similar amino acid substitutions for suchpredicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of a polypeptide withrespect to its three-dimensional structure. In certain embodiments, oneskilled in the art may choose to not make radical changes to amino acidresidues predicted to be on the surface of the polypeptide, since suchresidues may be involved in important interactions with other molecules.Moreover, one skilled in the art may generate test variants containing asingle amino acid substitution at each desired amino acid residue. Thevariants can then be screened using activity assays known to thoseskilled in the art. Such variants could be used to gather informationabout suitable variants. For example, if one discovered that a change toa particular amino acid residue resulted in destroyed, undesirablyreduced, or unsuitable activity, variants with such a change can beavoided. In other words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

The term “polypeptide fragment” and “truncated polypeptide” as usedherein refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion as compared to a corresponding full-lengthprotein. In certain embodiments, fragments can be, e.g., at least 5, atleast 10, at least 25, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 350, at least 400, at least450, at least 500, at least 600, at least 700, at least 800, at least900 or at least 1000 amino acids in length. In certain embodiments,fragments can also be, e.g., at most 1000, at most 900, at most 800, atmost 700, at most 600, at most 500, at most 450, at most 400, at most350, at most 300, at most 250, at most 200, at most 150, at most 100, atmost 50, at most 25, at most 10, or at most 5 amino acids in length. Afragment can further comprise, at either or both of its ends, one ormore additional amino acids, for example, a sequence of amino acids froma different naturally-occurring protein (e.g., an Fc or leucine zipperdomain) or an artificial amino acid sequence (e.g., an artificial linkersequence).

The terms “polypeptide variant” and “polypeptide mutant” as used hereinrefers to a polypeptide that comprises an amino acid sequence whereinone or more amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. In certain embodiments, the number of amino acid residues tobe inserted, deleted, or substituted can be, e.g., at least 1, at least2, at least 3, at least 4, at least 5, at least 10, at least 25, atleast 50, at least 75, at least 100, at least 125, at least 150, atleast 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 350, at least 400, at least 450 or at least 500amino acids in length. Variants of the present disclosure include fusionproteins.

A “derivative” of a polypeptide is a polypeptide that has beenchemically modified, e.g., conjugation to another chemical moiety suchas, for example, polyethylene glycol, albumin (e.g., human serumalbumin), phosphorylation, and glycosylation.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. In certainembodiments, the % identity is selected from, e.g., at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% or more sequence identity to agiven sequence. In certain embodiments, the % identity is in the rangeof, e.g., about 60% to about 70%, about 70% to about 80%, about 80% toabout 85%, about 85% to about 90%, about 90% to about 95%, or about 95%to about 99%.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. In certain embodiments, the % homology is selectedfrom, e.g., at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99% ormore sequence homology to a given sequence. In certain embodiments, the% homology is in the range of, e.g., about 60% to about 70%, about 70%to about 80%, about 80% to about 85%, about 85% to about 90%, about 90%to about 95%, or about 95% to about 99%.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the Gen Bank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.(Id).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci.USA, 90:5873-5787 (1993)). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is, e.g., less than about 0.1, less than about 0.01, orless than about 0.001.

The term “isolated molecule” (where the molecule is, for example, apolypeptide, a polynucleotide, or an antibody) is a molecule that byvirtue of its origin or source of derivation (1) is not associated withnaturally associated components that accompany it in its native state,(2) is substantially free of other molecules from the same species (3)is expressed by a cell from a different species, or (4) does not occurin nature. Thus, a molecule that is chemically synthesized, or expressedin a cellular system different from the cell from which it naturallyoriginates, will be “isolated” from its naturally associated components.A molecule also may be rendered substantially free of naturallyassociated components by isolation, using purification techniques wellknown in the art. Molecule purity or homogeneity may be assayed by anumber of means well known in the art. For example, the purity of apolypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60% to 75%of a sample exhibits a single species of polypeptide. A substantiallypure polypeptide or protein will typically comprise about 50%, 60%, 70%,80% or 90% W/W of a protein sample, more usually about 95%, and e.g.,will be over 99% pure. Protein purity or homogeneity may be indicated bya number of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein sample, followed by visualizing a singlepolypeptide band upon staining the gel with a stain well known in theart. For certain purposes, higher resolution may be provided by usingHPLC or other means well known in the art for purification.

An “antigen binding and antagonizing protein” is a protein comprising aportion that binds to an antigen and, optionally, a scaffold orframework portion that allows the antigen binding portion to adopt aconformation that promotes binding of the isolated antagonistic antigenbinding protein to the antigen. Examples of antigen binding andantagonizing proteins include antibodies, antibody fragments (e.g., anantigen binding portion of an antibody), antibody derivatives, andantibody analogs. The isolated antagonistic antigen binding protein cancomprise, for example, an alternative protein scaffold or artificialscaffold with grafted CDRs or CDR derivatives. Such scaffolds include,but are not limited to, antibody-derived scaffolds comprising mutationsintroduced to, for example, stabilize the three-dimensional structure ofthe isolated antagonistic antigen binding protein as well as whollysynthetic scaffolds comprising, for example, a biocompatible polymer.See, for example, Korndorfer et al., 2003, Proteins: Structure,Function, and Bioinformatics, Volume 53, Issue 1:121-129 (2003); Roqueet al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptideantibody mimetics (“PAMs”) can be used, as well as scaffolds based onantibody mimetics utilizing fibronection components as a scaffold.

An isolated antagonistic antigen binding protein can have, for example,the structure of a naturally occurring immunoglobulin. An“immunoglobulin” is a tetrameric molecule. In a naturally occurringimmunoglobulin, each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The amino-terminal portion of eachchain includes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminalportion of each chain defines a constant region primarily responsiblefor effector function. Human light chains are classified as kappa andlambda light chains. Heavy chains are classified as mu, delta, gamma,alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG,IgA, and IgE, respectively. Within light and heavy chains, the variableand constant regions are joined by a “J” region of about 12 or moreamino acids, with the heavy chain also including a “D” region of about10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul,W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference inits entirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site such that an intactimmunoglobulin has two binding sites.

An “antibody” refers to a protein comprising one or more polypeptidessubstantially or partially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes and having specificity to a tumor antigen orspecificity to a molecule overexpressed in a pathological state. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as subtypes ofthese genes and myriad of immunoglobulin variable region genes. Lightchains (LC) are classified as either kappa or lambda. Heavy chains (HC)are classified as gamma, mu, alpha, delta, or epsilon, which in turndefine the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (e.g., antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or V_(H)) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, C_(H1), C_(H2) and C_(H3) (and in some instances,C_(H4)). Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or V_(L)) and a light chain constant region.The light chain constant region is comprised of one domain, C_(L). TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR₁, CDR₁, FR₂, CDR₂, FR₃, CDR₃, FR₄. The extent of the frameworkregion and CDRs has been defined. The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies, such as humans. The framework region of an antibody, that isthe combined framework regions of the constituent light and heavychains, serves to position and align the CDRs in three-dimensionalspace. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1 and IgA2)or subclass.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments. Such fragments include Fab fragments, Fab′fragments, Fab₂, F(ab)′₂ fragments, single chain Fv proteins (“scFv”)and disulfide stabilized Fv proteins (“dsFv”), that bind to the targetantigen. A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, as usedherein, the term antibody encompasses e.g., monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, human antibodies, humanized antibodies,camelised antibodies, chimeric antibodies, single-chain Fvs (scFv),single-chain antibodies, single domain antibodies, domain antibodies,Fab fragments, F(ab′)₂ fragments, antibody fragments that exhibit thedesired biological activity, disulfide-linked Fvs (sdFv), intrabodies,and epitope-binding fragments or antigen binding fragments of any of theabove.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C.sub.H1 domains; an Fv fragment has theV_(L) and V_(H) domains of a single arm of an antibody; and a dAbfragment has a V_(H) domain, a V_(L) domain, or an antigen-bindingfragment of a V_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634,6,696,245, US App. Pub. No. 05/0202512, 04/0202995, 04/0038291,04/0009507, 03/0039958, Ward et al., Nature 341:544-546 (1989)).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,Science 242:423-26 (1988) and Huston et al., 1988, Proc. Natl. Acad.Sci. USA 85:5879-83 (1988)). Diabodies are bivalent antibodiescomprising two polypeptide chains, wherein each polypeptide chaincomprises V_(H) and V_(L) domains joined by a linker that is too shortto allow for pairing between two domains on the same chain, thusallowing each domain to pair with a complementary domain on anotherpolypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad.Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2:1121-23(1994)). If the two polypeptide chains of a diabody are identical, thena diabody resulting from their pairing will have two identical antigenbinding sites. Polypeptide chains having different sequences can be usedto make a diabody with two different antigen binding sites. Similarly,tribodies and tetrabodies are antibodies comprising three and fourpolypeptide chains, respectively, and forming three and four antigenbinding sites, respectively, which can be the same or different.

An isolated antagonistic antigen binding protein may have one or morebinding sites. If there is more than one binding site, the binding sitesmay be identical to one another or may be different. For example, anaturally occurring human immunoglobulin typically has two identicalbinding sites, while a “bispecific” or “bifunctional” antibody has twodifferent binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A “humanized antibody” has a sequence that differs from the sequence ofan antibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

An isolated antagonistic antigen binding protein of the presentdisclosure, including an antibody, “specifically binds” to an antigen,such as the human glucagon receptor if it binds to the antigen with ahigh binding affinity as determined by a dissociation constant (Kd, orcorresponding Kb, as defined below) value of 10⁻⁷ M or less. An isolatedantagonistic antigen binding protein that specifically binds to thehuman glucagon receptor may be able to bind to glucagon receptors fromother species as well with the same or different affinities.

An “epitope” is the portion of a molecule that is bound by an isolatedantagonistic antigen binding protein (e.g., by an antibody). An epitopecan comprise non-contiguous portions of the molecule (e.g., in apolypeptide, amino acid residues that are not contiguous in thepolypeptide's primary sequence but that, in the context of thepolypeptide's tertiary and quaternary structure, are near enough to eachother to be bound by an antigen binding and antagonizing protein).

The term “blood glucose level”, or “level of blood glucose” shall meanblood glucose concentration. In certain embodiments, a blood glucoselevel is a plasma glucose level. Plasma glucose may be determined inaccordance with, e.g., Etgen et al., Metabolism, 49(5): 684-688, 2000)or calculated from a conversion of whole blood glucose concentration inaccordance with D'Orazio et al., Clin. Chem. Lab. Med.,44(12):1486-1490, 2006.

The term “normal glucose levels” refers to mean plasma glucose values inhumans of less than about 100 mg/dL for fasting levels, and less thanabout 145 mg/dL for 2-hour post-prandial levels or 125 mg/dL for arandom glucose. The term “elevated blood glucose level” or “elevatedlevels of blood glucose” shall mean an elevated blood glucose level suchas that found in a subject demonstrating clinically inappropriate basaland postprandial hyperglycemia or such as that found in a subject inoral glucose tolerance test (oGTT), with “elevated levels of bloodglucose” being greater than about 100 mg/dL when tested under fastingconditions, and greater than about 200 mg/dL when tested at 1 hour.

The terms “glucagon inhibitor”, “glucagon suppressor” and “glucagonantagonist” are used interchangeably. Each is a molecule that detectablyinhibits glucagon signaling. The inhibition caused by an inhibitor neednot be complete so long as the inhibition is detectable using an assaythat is recognized and understood in the art as being determinative ofglucagon signaling inhibition.

As used herein, “heart failure” means an abnormality of cardiac functionwhere the heart does not pump blood at the rate needed for therequirements of metabolizing tissues. Heart failure includes a widerange of disease states such as congestive heart failure, myocardialinfarction, tachyarrhythmia, familial hypertrophic cardiomyopathy,ischemic heart disease, idiopathic dilated cardiomyopathy, myocarditisand the like. The heart failure can be caused by any number of factors,including, without limitation, ischemic, congenital, rheumatic, viral,toxic or idiopathic forms. Chronic cardiac hypertrophy is asignificantly diseased state which is a precursor to congestive heartfailure and cardiac arrest.

A “pharmaceutical composition” refers to a composition suitable forpharmaceutical use in an animal or human. A pharmaceutical compositioncomprises a pharmacologically and/or therapeutically effective amount ofan active agent and a pharmaceutically acceptable carrier.“Pharmaceutically acceptable carrier” refers to compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein “pharmaceutically acceptablecarrier” refers to any of the standard pharmaceutical carriers,vehicles, buffers, and carriers, such as a phosphate buffered salinesolution, 5% aqueous solution of dextrose, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agentsand/or adjuvants. Suitable pharmaceutical carriers and formulations aredescribed in Remington's Pharmaceutical Sciences, 21st Ed. 2005, MackPublishing Co, Easton. A “pharmaceutically acceptable salt” is a saltthat can be formulated into a compound for pharmaceutical use including,e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines.

The term “immunoconjugate” or “fusion protein” as used herein refers toa molecule comprising an antibody or antigen-binding fragment thereofconjugated (or linked) directly or indirectly to an effector molecule.The effector molecule can be a detectable label, an immunotoxin,cytokine, chemokine, therapeutic agent, or chemotherapeutic agent. Theantibody or antigen-binding fragment thereof may be conjugated to aneffector molecule via a peptide linker. An immunoconjugate and/or fusionprotein retains the immunoreactivity of the antibody or antigen-bindingfragment, e.g., the antibody or antigen-binding fragment hasapproximately the same, or only slightly reduced, ability to bind theantigen after conjugation as before conjugation. As used herein, animmunoconjugate may also be referred to as an antibody drug conjugate(ADC). Because immunoconjugates and/or fusion proteins are originallyprepared from two molecules with separate functionalities, such as anantibody and an effector molecule, they are also sometimes referred toas “chimeric molecules.”

As used herein, a “therapeutically effective amount” of an isolatedantagonistic antigen binding protein that specifically binds the humanglucagon receptor refers to an amount of such protein that, whenprovided to a subject in accordance with the disclosed and claimedmethods effects one of the following biological activities: treats heartfailure; or reduces, suppresses, attenuates, or inhibits one or moresymptoms of heart failure.

The terms “treat”, “treating” and “treatment” refer refers to anapproach for obtaining beneficial or desired clinical results. Further,references herein to “treatment” include references to curative,palliative and prophylactic treatment. For purposes of this disclosure,beneficial or desired clinical results include, but are not limited to,one or more of the following: improvement in blood glucose to withinabout 80-180 mg/dL, or to within about 80-170 mg/dL, or to within about80-160 mg/dL, or to within about 80-150 mg/dL, or to within about 80-140mg/dL, or an improvement in any one or more conditions, diseases, orsymptoms associated with, or resulting from, elevated levels of bloodglucose including, but not limited to, hyperglycemia, hyperglucanemia,and hyperinsulinemia.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that aspects and variations of thedisclosure described herein include “consisting” and/or “consistingessentially of” aspects and variation.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

Glucagon Receptor and Antigen Binding and Antagonizing Proteins

Glucagon is a 29 amino acid hormone processed from its pre-pro-form inthe pancreatic alpha cells by cell specific expression of prohormoneconvertase 2 (PC2), a neuroendocrine-specific protease involved in theintracellular maturation of prohormones and proneuropeptides (Furuta etal., J. Biol. Chem. 276: 27197-27202 (2001)). In vivo, glucagon is amajor counter-regulatory hormone for insulin actions. During fasting,glucagon secretion increases in response to falling glucose levels.Increased glucagon secretion stimulates glucose production by promotinghepatic glycogenolysis and gluconeogenesis (Dunning and Gerich,Endocrine Reviews, 28:253-283 (2007)). Thus glucagon counterbalances theeffects of insulin in maintaining normal levels of glucose in animals.

The biological effects of glucagon are mediated through the binding andsubsequent activation of a specific cell surface receptor, the glucagonreceptor. The glucagon receptor (GCGR) is a member of the secretinsubfamily (family B) of G-protein-coupled receptors. The human GCGR is a477 amino acid sequence GPCR and the amino acid sequence of GCGR ishighly conserved across species (Mayo et al, Pharmacological Rev.,55:167-194, (2003)). The glucagon receptor is predominantly expressed inthe liver, where it regulates hepatic glucose output, on the kidney, andon islet β-cells, reflecting its role in gluconeogenesis. The activationof the glucagon receptors in the liver stimulates the activity of adenylcyclase and phosphoinositol turnover which subsequently results inincreased expression of gluconeogenic enzymes includingphosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase(FBPase-1), and glucose-6-phosphatase (G-6-Pase). In addition, glucagonsignaling activates glycogen phosphorylase and inhibits glycogensynthase. Studies have shown that higher basal glucagon levels and lackof suppression of postprandial glucagon secretion contribute to diabeticconditions in humans (Muller et al., N Eng J Med 283: 109-115 (1970)).As such, methods of controlling and lowering blood glucose by targetingglucagon production or function using a GCGR antagonist have beenexplored.

In various embodiments, the antigen binding and antagonizing proteins ofthe present disclosure may be selected to bind to membrane-boundglucagon receptors as expressed on cells, and inhibit or block glucagonsignaling through the glucagon receptor. In various embodiments, theantigen binding and antagonizing proteins of the present disclosurespecifically bind to the human glucagon receptor. In variousembodiments, the antigen binding and antagonizing proteins binding tothe human glucagon receptor may also bind to the glucagon receptors ofother species. The polynucleotide and polypeptide sequences for severalspecies of glucagon receptor are known (see, e.g., U.S. Pat. No.7,947,809, herein incorporated by reference in its entirety for itsspecific teaching of polynucleotide and polypeptide sequences of ahuman, rat, mouse and cynomolgus glucagon receptor). In variousembodiments of the present disclosure, the antigen binding andantagonizing proteins specifically bind the human glucagon receptorhaving the amino acid sequence set forth in SEQ ID NO: 1:

Glucagon Receptor Human (Homo sapiens)amino acid sequence (Accession Number AAI04855) (SEQ ID NO: 1)MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQPWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPFIn various embodiments, the antigen binding and antagonizing proteins ofthe present disclosure specifically bind glucagon receptors which haveat least 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity (as calculated using methods known in the art and describedherein) to the glucagon receptors described in the cited references arealso included in the present disclosure.

The antigen binding and antagonizing proteins of the present disclosurefunction to block the interaction between glucagon and its receptor,thereby inhibiting the glucose elevating effects of glucagon. As such,the use of the antigen binding and antagonizing proteins of the presentdisclosure are an effective means of achieving normal levels of glucose,thereby ameliorating, or preventing one or more symptoms of, or longterm complications caused by diabetes including, but not limited to,hyperglycemia, hyperglucanemia, and hyperinsulinemia. The use of theantigen binding and antagonizing proteins of the present disclosure arealso an effective means of achieving normal levels of glucose innon-diabetic patients, thereby lowering the risk of hyperglycemia,hyperglucanemia, and hyperinsulinemia in subjects having disordersincluding, but not limited to, T1 D, T2D, obesity, NAFLD, NASH, and fortreating such non-diabetic disorders. The present disclosure is based inpart on the inventors' unique insight that isolated antigen binding andantagonizing proteins that specifically bind to the human glucagonreceptor may provide for improved, effective therapies for treatment andprevention of heart failure after myocardial infarction. The presentinventor proposes that the beneficial therapeutic effects provided byblocking the glucagon receptor in myocardial infarction-induced heartfailure subjects (or all other heart failure subjects) relate to theprevention or attenuation of left ventricular (LV) remodeling which mayinclude: increasing fractional shortening (FS), decreasing LV dilation(LVESD); preserving LV wall thickness; increasing LV developed pressureand ventricular contractility; decreasing the ratio of heart weight tobody weight (infarct size); reducing the fibrosis process; reducinglevels of incompletely oxidized fatty acid metabolites in the heartunder ischemic conditions; and reducing MI-induced mortality rate.

Methods of generating antibodies that bind to antigens such as the humanglucagon receptor are known to those skilled in the art. For example, amethod for generating a monoclonal antibody that binds specifically to atargeted antigen polypeptide may comprise administering to a mouse anamount of an immunogenic composition comprising the targeted antigenpolypeptide effective to stimulate a detectable immune response,obtaining antibody-producing cells (e.g., cells from the spleen) fromthe mouse and fusing the antibody-producing cells with myeloma cells toobtain antibody-producing hybridomas, and testing the antibody-producinghybridomas to identify a hybridoma that produces a monocolonal antibodythat binds specifically to the targeted antigen polypeptide. Onceobtained, a hybridoma can be propagated in a cell culture, optionally inculture conditions where the hybridoma-derived cells produce themonoclonal antibody that binds specifically to targeted antigenpolypeptide. The monoclonal antibody may be purified from the cellculture. A variety of different techniques are then available fortesting an antigen/antibody interaction to identify particularlydesirable antibodies.

Other suitable methods of producing or isolating antibodies of therequisite specificity can used, including, for example, methods whichselect recombinant antibody from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies. See e.g., Jakobovits et al., Proc.Natl. Acad. Sci. (U.S.A.), 90: 2551-2555, 1993; Jakobovits et al.,Nature, 362: 255-258, 1993; Lonberg et al., U.S. Pat. No. 5,545,806; andSurani et al., U.S. Pat. No. 5,545,807.

Antibodies can be engineered in numerous ways. They can be made assingle-chain antibodies (including small modular immunopharmaceuticalsor SMIPs™), Fab and F(ab′)2 fragments, etc. Antibodies can be humanized,chimerized, deimmunized, or fully human. Numerous publications set forththe many types of antibodies and the methods of engineering suchantibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370;5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and5,260,203.

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art. For example, a gene encoding the Fc constant region of amurine (or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al., Science,240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci. (U.S.A.),84:3439-3443, 1987; Liu et al., J. Immunol., 139:3521-3526, 1987; Sun etal., Proc. Natl. Acad. Sci. (U.S.A.), 84:214-218, 1987; Nishimura etal., Canc. Res., 47:999-1005, 1987; Wood et al., Nature, 314:446-449,1985; and Shaw et al., J. Natl Cancer Inst., 80:1553-1559, 1988).

Methods for humanizing antibodies have been described in the art. Insome embodiments, a humanized antibody has one or more amino acidresidues introduced from a source that is nonhuman, in addition to thenonhuman CDRs. Humanization can be essentially performed following themethod of Winter and co-workers (Jones et al., Nature, 321:522-525,1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al.,Science, 239:1534-1536, 1988), by substituting hypervariable regionsequences for the corresponding sequences of a human antibody.Accordingly, such “humanized” antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567) wherein substantially less than an intact humanvariable region has been substituted by the corresponding sequence froma nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some hypervariable region residues andpossibly some framework region residues are substituted by residues fromanalogous sites in rodent antibodies.

U.S. Pat. No. 5,693,761 to Queen et al, discloses a refinement on Winteret al. for humanizing antibodies, and is based on the premise thatascribes avidity loss to problems in the structural motifs in thehumanized framework which, because of steric or other chemicalincompatibility, interfere with the folding of the CDRs into thebinding-capable conformation found in the mouse antibody. To addressthis problem, Queen teaches using human framework sequences closelyhomologous in linear peptide sequence to framework sequences of themouse antibody to be humanized. Accordingly, the methods of Queen focuson comparing framework sequences between species. Typically, allavailable human variable region sequences are compared to a particularmouse sequence and the percentage identity between correspondentframework residues is calculated. The human variable region with thehighest percentage is selected to provide the framework sequences forthe humanizing project. Queen also teaches that it is important toretain in the humanized framework, certain amino acid residues from themouse framework critical for supporting the CDRs in a binding-capableconformation. Potential criticality is assessed from molecular models.Candidate residues for retention are typically those adjacent in linearsequence to a CDR or physically within 6 Å of any CDR residue.

In other approaches, the importance of particular framework amino acidresidues is determined experimentally once a low-avidity humanizedconstruct is obtained, by reversion of single residues to the mousesequence and assaying antigen binding as described by Riechmann et al,1988. Another example approach for identifying important amino acids inframework sequences is disclosed by U.S. Pat. No. 5,821,337 to Carter etal, and by U.S. Pat. No. 5,859,205 to Adair et al. These referencesdisclose specific Kabat residue positions in the framework, which, in ahumanized antibody may require substitution with the correspondent mouseamino acid to preserve avidity.

Another method of humanizing antibodies, referred to as “frameworkshuffling”, relies on generating a combinatorial library with nonhumanCDR variable regions fused in frame into a pool of individual humangermline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). Thelibraries are then screened to identify clones that encode humanizedantibodies which retain good binding.

The choice of human variable regions, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable region of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (framework region) for the humanized antibody(Sims et al., J. Immunol., 151:2296, 1993; Chothia et al., J. Mol.Biol., 196:901, 1987). Another method uses a particular framework regionderived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chain variable regions. The sameframework may be used for several different humanized antibodies (Carteret al., Proc. Natl. Acad. Sci. (U.S.A.), 89:4285, 1992; Presta et al.,J. Immunol., 151:2623, 1993).

The choice of nonhuman residues to substitute into the human variableregion can be influenced by a variety of factors. These factors include,for example, the rarity of the amino acid in a particular position, theprobability of interaction with either the CDRs or the antigen, and theprobability of participating in the interface between the light andheavy chain variable domain interface. (See, for example, U.S. Pat. Nos.5,693,761, 6,632,927, and 6,639,055). One method to analyze thesefactors is through the use of three-dimensional models of the nonhumanand humanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available that illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, e.g., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, nonhuman residues can be selected and substituted for humanvariable region residues in order to achieve the desired antibodycharacteristic, such as increased affinity for the target antigen(s).

Methods for making fully human antibodies have been described in theart. By way of example, a method for producing an anti-GCGR antibody orantigen binding antibody fragment thereof comprises the steps ofsynthesizing a library of human antibodies on phage, screening thelibrary with GCGR or an antibody binding portion thereof, isolatingphage that bind GCGR, and obtaining the antibody from the phage. By wayof another example, one method for preparing the library of antibodiesfor use in phage display techniques comprises the steps of immunizing anon-human animal comprising human immunoglobulin loci with GCGR or anantigenic portion thereof to create an immune response, extractingantibody-producing cells from the immunized animal; isolating RNAencoding heavy and light chains of antibodies of the disclosure from theextracted cells, reverse transcribing the RNA to produce cDNA,amplifying the cDNA using primers, and inserting the cDNA into a phagedisplay vector such that antibodies are expressed on the phage.Recombinant anti-GCGR antibodies of the disclosure may be obtained inthis way.

Again, by way of example, recombinant human anti-GCGR antibodies of thedisclosure can also be isolated by screening a recombinant combinatorialantibody library. Preferably the library is a scFv phage displaylibrary, generated using human V_(L) and V_(H) cDNAs prepared from mRNAisolated from B cells. Methods for preparing and screening suchlibraries are known in the art. Kits for generating phage displaylibraries are commercially available (e.g., the Pharmacia RecombinantPhage Antibody System, catalog no. 27-9400-01; and the StratageneSurfZAP™ phage display kit, catalog no. 240612). There also are othermethods and reagents that can be used in generating and screeningantibody display libraries (see, e.g., U.S. Pat. No. 5,223,409; PCTPublication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio/Technology,9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas, 3:81-85, 1992;Huse et al., Science, 246:1275-1281, 1989; McCafferty et al., Nature,348:552-554, 1990; Griffiths et al., EMBO J., 12:725-734, 1993; Hawkinset al., J. Mol. Biol., 226:889-896, 1992; Clackson et al., Nature,352:624-628, 1991; Gram et al., Proc. Natl. Acad. Sci. (U.S.A.),89:3576-3580, 1992; Garrad et al., Bio/Technology, 9:1373-1377, 1991;Hoogenboom et al., Nuc. Acid Res., 19:4133-4137, 1991; and Barbas etal., Proc. Natl. Acad. Sci. (U.S.A.), 88:7978-7982, 1991), allincorporated herein by reference.

Human antibodies are also produced by immunizing a non-human, transgenicanimal comprising within its genome some or all of human immunoglobulinheavy chain and light chain loci with a human IgE antigen, e.g., aXenoMouse™ animal (Abgenix, Inc./Amgen, Inc.—Fremont, Calif.).XenoMouse™ mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics, 7:13-21, 1994 and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. XenoMouse™ mice produce an adult-like humanrepertoire of fully human antibodies and generate antigen-specific humanantibodies. In some embodiments, the XenoMouse™ mice containapproximately 80% of the human antibody V gene repertoire throughintroduction of megabase sized, germline configuration fragments of thehuman heavy chain loci and kappa light chain loci in yeast artificialchromosome (YAC). In other embodiments, XenoMouse™ mice further containapproximately all of the human lambda light chain locus. See Mendez etal., Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J. Exp.Med., 188:483-495, 1998; and WO 98/24893.

In various embodiments, the isolated antagonistic antigen bindingprotein of the present disclosure utilize an antibody or antigen bindingantibody fragment thereof is a polyclonal antibody, a monoclonalantibody or antigen-binding fragment thereof, a recombinant antibody, adiabody, a chimerized or chimeric antibody or antigen-binding fragmentthereof, a humanized antibody or antigen-binding fragment thereof, afully human antibody or antigen-binding fragment thereof, a CDR-graftedantibody or antigen-binding fragment thereof, a single chain antibody,an Fv, an Fd, an Fab, an Fab′, or an F(ab′)₂, and synthetic orsemi-synthetic antibodies.

In various embodiments, the isolated antagonistic antigen bindingprotein of the present disclosure utilize an antibody or antigen-bindingfragment that binds to an immune-checkpoint protein antigen with adissociation constant (K_(D)) of, e.g., at least about 1×10⁻⁷ M, atleast about 1×10⁻⁸ M, at least about 1×10⁻⁸ M, at least about 1×10⁻¹⁰M,at least about 1×10⁻¹¹ M, or at least about 1×10⁻¹² M. In variousembodiments, the isolated antagonistic antigen binding protein of thepresent disclosure utilize an antibody or antigen-binding fragment thatbinds to an immune-checkpoint protein antigen with a dissociationconstant (K_(D)) in the range of, e.g., at least about 1×10⁻⁷ M to atleast about 1×10⁻⁸ M, at least about 1×10⁻⁸ M to at least about 1×10⁻⁸M, at least about 1×10⁻⁸ M to at least about 1×10⁻¹⁰M, at least about1×10⁻¹⁰ M to at least about 1×10⁻¹¹ M, or at least about 1×10⁻¹¹ M to atleast about 1×10⁻¹² M.

In various embodiments of the present invention, the isolatedantagonistic antigen binding protein is an anti-GCGR (“antagonistic”)antibody or antigen-binding fragment which comprises the polynucleotideand polypeptide sequences set forth in, e.g., U.S. Pat. Nos. 7,947,809,8,158,759, 5,770,445, 7,947,809, 7,968,686, 8,545,847, and 8,771,696;U.S. patent publications 2009/0041784; 2009/0252727; 2013/0344538 and2014/0335091; and PCT publication WO2008/036341, each hereinincorporated by reference in its entirety for its specific teaching ofpolynucleotide and polypeptide sequences of various anti-GCGR antibodiesor antigen-binding fragments. In various embodiments of the presentinvention, the isolated antagonistic antigen binding protein is anybiosimilar, biogeneric, follow-on biologic, or follow-on protein versionof any anti-GCGR antibody described in the art.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the heavy chain variable regionsequence as set forth in SEQ ID NO: 2. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the heavy chain variable region sequence as setforth in SEQ ID NO: 2. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the heavychain variable region sequence as set forth in SEQ ID NO: 2. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the heavy chain variableregion sequence as set forth in SEQ ID NO: 2. In various embodiments,the antibody may be an anti-GCGR antibody having a sequence identical,substantially identical or substantially similar to SEQ ID NO: 2. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the heavy chain variable region sequence as set forth in SEQID NO: 2:

(SEQ ID NO: 2) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYN YYYGLDVWGQGTTVTVSS

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the light chain variable regionsequence as set forth in SEQ ID NO: 3. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the light chain variable region sequence as setforth in SEQ ID NO: 3. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the lightchain variable region sequence as set forth in SEQ ID NO: 3. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the light chain variableregion sequence as set forth in SEQ ID NO: 3. In various embodiments,the antibody may be an anti-GCGR antibody having a sequence identical,substantially identical or substantially similar to SEQ ID NO: 3. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the light chain variable region sequence as set forth in SEQID NO: 3:

(SEQ ID NO: 3) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGG GTKVEIK

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 2 or 3.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the heavy chain variable regionsequence as set forth in SEQ ID NO: 4. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the heavy chain variable region sequence as setforth in SEQ ID NO: 4. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the heavychain variable region sequence as set forth in SEQ ID NO: 4. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the heavy chain variableregion sequence as set forth in SEQ ID NO: 4. In various embodiments,the antibody may be an anti-GCGR antibody having a sequence identical,substantially identical or substantially similar to SEQ ID NO: 4. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the heavy chain variable region sequence as set forth in SEQID NO: 4:

(SEQ ID NO: 4) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSS

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the light chain variable regionsequence as set forth in SEQ ID NO: 5. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the light chain variable region sequence as setforth in SEQ ID NO: 5. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the lightchain variable region sequence as set forth in SEQ ID NO: 5. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the light chain variableregion sequence as set forth in SEQ ID NO: 5. In various embodiments,the antibody may be an anti-GCGR antibody having a sequence identical,substantially identical or substantially similar to SEQ ID NO: 5. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the light chain variable region sequence as set forth in SEQID NO: 5:

(SEQ ID NO: 5) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFVTYYCLQHNSNPLTFGG GTKVEIK

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 4 or 5.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the heavy chain variable regionsequence as set forth in SEQ ID NO: 6. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the heavy chain variable region sequence as setforth in SEQ ID NO: 6. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the heavychain variable region sequence as set forth in SEQ ID NO: 6. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the heavy chain variableregion sequence as set forth in SEQ ID NO: 6. In various embodiments,the antibody may be an anti-GCGR antibody having a sequence identical,substantially identical or substantially similar to SEQ ID NO: 6. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the heavy chain variable region sequence as set forth in SEQID NO: 6:

(SEQ ID NO: 6) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSS

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the light chain variable regionsequence as set forth in SEQ ID NO: 7. In various embodiments, theantibody may be an anti-GCGR antibody which binds to the same epitope asthe antibody comprising the light chain variable region sequence as setforth in SEQ ID NO: 7. In various embodiments, the antibody is ananti-GCGR antibody which competes with the antibody comprising the lightchain variable region sequence as set forth in SEQ ID NO: 7. In variousembodiments, the antibody may be an anti-GCGR antibody which comprisesat least one (such as two or three) CDRs of the light chain variableregion sequence as set forth in SEQ ID NO: 7. In various embodiments,the antibody may be an anti-GCGR antibody having a sequence identical,substantially identical or substantially similar to SEQ ID NO: 7. Invarious embodiments, the antibody may be an anti-GCGR antibody whichcomprises the light chain variable region sequence as set forth in SEQID NO: 7:

(SEQ ID NO: 7) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGG GTKVEIK

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 6 or 7.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the chimeric antibody comprising the heavy chain sequence asset forth in SEQ ID NO: 8. In various embodiments, the antibody may bean anti-GCGR antibody which binds to the same epitope as the antibodycomprising the heavy chain sequence as set forth in SEQ ID NO: 8. Invarious embodiments, the antibody is an anti-GCGR antibody whichcompetes with the antibody comprising the heavy chain sequence as setforth in SEQ ID NO: 8. In various embodiments, the antibody may be ananti-GCGR antibody which comprises at least one (such as two or three)CDRs of the heavy chain sequence as set forth in SEQ ID NO: 8. Invarious embodiments, the antibody may be an anti-GCGR antibody having asequence identical, substantially identical or substantially similar toSEQ ID NO: 8. In various embodiments, the antibody may be an anti-GCGRantibody which comprises the heavy chain sequence as set forth in SEQ IDNO: 8:

(SEQ ID NO: 8) MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGK

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody that has the same or higher antigen-binding affinityas that of the chimeric antibody comprising the light chain sequence asset forth in SEQ ID NO: 9. In various embodiments, the antibody may bean anti-GCGR antibody which binds to the same epitope as the antibodycomprising the light chain sequence as set forth in SEQ ID NO: 9. Invarious embodiments, the antibody is an anti-GCGR antibody whichcompetes with the antibody comprising the light chain sequence as setforth in SEQ ID NO: 9. In various embodiments, the antibody may be ananti-GCGR antibody which comprises at least one (such as two or three)CDRs of the light chain sequence as set forth in SEQ ID NO: 9. Invarious embodiments, the antibody may be an anti-GCGR antibody having asequence identical, substantially identical or substantially similar toSEQ ID NO: 9. In various embodiments, the antibody may be an anti-GCGRantibody which comprises the light chain sequence as set forth in SEQ IDNO: 9:

(SEQ ID NO: 9) MDMRVPAQLLGLLLLWFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

In various embodiments, the antibody contains an amino acid sequencethat shares an observed homology of, e.g., at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the sequences of SEQ IDNOS: 8 or 9.

In various embodiments of the present disclosure the antibody may be ananti-GCGR antibody which comprises a heavy chain variable regionsequence identical, substantially identical or substantially similar toa sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQID NO: 27, and SEQ ID NO: 28 and a light chain variable region sequenceidentical, substantially identical or substantially similar to asequence selected from SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ IDNO: 46, and SEQ ID NO: 47. In various embodiments, the antibody containsan amino acid sequence that shares an observed homology of, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%with the sequences of SEQ ID NOS: 10-28 or SEQ ID NOS: 29-47.

Examples of Anti-GCGR Antibodies

HCVR LCVR SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO:6 SEQ ID NO: 7 SEQ ID NO: 10 SEQ ID NO: 29 SEQ ID NO: 11 SEQ ID NO: 30SEQ ID NO: 12 SEQ ID NO: 31 SEQ ID NO: 13 SEQ ID NO: 32 SEQ ID NO: 14SEQ ID NO: 33 SEQ ID NO: 15 SEQ ID NO: 34 SEQ ID NO: 16 SEQ ID NO: 35SEQ ID NO: 17 SEQ ID NO: 36 SEQ ID NO: 18 SEQ ID NO: 37 SEQ ID NO: 19SEQ ID NO: 38 SEQ ID NO: 20 SEQ ID NO: 39 SEQ ID NO: 21 SEQ ID NO: 40SEQ ID NO: 22 SEQ ID NO: 41 SEQ ID NO: 23 SEQ ID NO: 42 SEQ ID NO: 24SEQ ID NO: 43 SEQ ID NO: 25 SEQ ID NO: 44 SEQ ID NO: 26 SEQ ID NO: 45SEQ ID NO: 27 SEQ ID NO: 46 SEQ ID NO: 28 SEQ ID NO: 47

An isolated anti-GCGR antibody, antibody fragment, or antibodyderivative of the present disclosure can comprise any constant regionknown in the art. The light chain constant region can be, for example, akappa- or lambda-type light chain constant region, e.g., a human kappa-or lambda-type light chain constant region. The heavy chain constantregion can be, for example, an alpha-, delta-, epsilon-, gamma-, ormu-type heavy chain constant regions, e.g., a human alpha-, delta-,epsilon-, gamma-, or mu-type heavy chain constant region. In variousembodiments, the light or heavy chain constant region is a fragment,derivative, variant, or mutein of a naturally occurring constant region.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype. See also Lanitto et al., Methods Mol. Biol. 178:303-16 (2002).

In various embodiments, an isolated antigen binding protein of thepresent disclosure comprises the constant light chain kappa region asset forth in SEQ ID NO: 48, or a fragment thereof. In variousembodiments, an isolated antigen binding protein of the presentdisclosure comprises the constant light chain lambda region as set forthin SEQ ID NO: 49, or a fragment thereof. In various embodiments, anisolated antigen binding protein of the present disclosure comprises aIgG2 heavy chain constant region set forth in SEQ ID NO: 50, or afragment thereof.

In various embodiments, an isolated antagonistic antigen binding proteinof the present disclosure is a fully human anti-GCGR antibody thatcomprises a heavy chain sequence identical, substantially identical orsubstantially similar to SEQ ID NO: 51 and a light chain sequenceidentical, substantially identical or substantially similar to SEQ IDNO: 52. In various embodiments, an isolated antagonistic antigen bindingprotein of the present disclosure is a fully human anti-GCGR antibodythat comprises a heavy chain sequence as set forth in SEQ ID NO: 51 anda light chain as set forth in SEQ ID NO: 52.

In various embodiments of the present disclosure, the isolatedantagonistic antigen binding protein is a hemibody. A “hemibody” is animmunologically-functional immunoglobulin construct comprising acomplete heavy chain, a complete light chain and a second heavy chain Fcregion paired with the Fc region of the complete heavy chain. A linkercan, but need not, be employed to join the heavy chain Fc region and thesecond heavy chain Fc region. In various embodiments, the hemibody is amonovalent antigen binding protein comprising (i) an intact light chain,and (ii) a heavy chain fused to an Fc region (e.g., an IgG2 Fc region).Methods for preparing hemibodies are described in, e.g., U.S. patentapplication 2012/0195879, herein incorporated by reference in itsentirety herein for purposes of teaching the preparation of suchhemibodies.

Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising an isolated antagonistic antigen binding proteinas described herein, with one or more pharmaceutically acceptablecarrier(s). The pharmaceutical compositions and methods of usesdescribed herein also encompass embodiments of combinations(co-administration) with other active agents, as detailed below.

Generally, the antagonistic antigen binding proteins of the presentdisclosure are suitable to be administered as a formulation inassociation with one or more pharmaceutically acceptable carrier(s). Theterm ‘carrier’ is used herein to describe any ingredient other than thecompound(s) of the disclosure. The choice of carrier(s) will to a largeextent depend on factors such as the particular mode of administration,the effect of the carrier on solubility and stability, and the nature ofthe dosage form. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. Some examples ofpharmaceutically acceptable carriers are water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, the composition will includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Additional examples ofpharmaceutically acceptable substances are wetting agents or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody. Pharmaceutical compositions of the present disclosureand methods for their preparation will be readily apparent to thoseskilled in the art. Such compositions and methods for their preparationmay be found, for example, in Remington's Pharmaceutical Sciences, 19thEdition (Mack Publishing Company, 1995). The pharmaceutical compositionsare generally formulated as sterile, substantially isotonic and in fullcompliance with all GMP regulations of the U.S. Food and DrugAdministration.

The pharmaceutical compositions of the present disclosure are typicallysuitable for parenteral administration. As used herein, “parenteraladministration” of a pharmaceutical composition includes any route ofadministration characterized by physical breaching of a tissue of asubject and administration of the pharmaceutical composition through thebreach in the tissue, thus generally resulting in the directadministration into the blood stream, into muscle, or into an internalorgan. Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous injection, intraperitoneal injection, intramuscularinjection, intrasternal injection, intravenous injection, intraarterialinjection, intrathecal injection, intraventricular injection,intraurethral injection, intracranial injection, intrasynovial injectionor infusions; or kidney dialytic infusion techniques.

A pharmaceutical composition of the present disclosure can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present disclosure. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded. Numerous reusable pen and autoinjector deliverydevices have been described in the literature and are commerciallyavailable.

Formulations of a pharmaceutical composition suitable for parenteraladministration typically generally comprise the active ingredientcombined with a pharmaceutically acceptable carrier, such as sterilewater or sterile isotonic saline. Such formulations may be prepared,packaged, or sold in a form suitable for bolus administration or forcontinuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampoules or inmulti-dose containers containing a preservative. Formulations forparenteral administration include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, and the like.Such formulations may further comprise one or more additionalingredients including, but not limited to, suspending, stabilizing, ordispersing agents. In one embodiment of a formulation for parenteraladministration, the active ingredient is provided in dry (i.e. powder orgranular) form for reconstitution with a suitable vehicle (e.g. sterilepyrogen-free water) prior to parenteral administration of thereconstituted composition. Parenteral formulations also include aqueoussolutions which may contain carriers such as salts, carbohydrates andbuffering agents (preferably to a pH of from 3 to 9), but, for someapplications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water. Exemplaryparenteral administration forms include solutions or suspensions insterile aqueous solutions, for example, aqueous propylene glycol ordextrose solutions. Such dosage forms can be suitably buffered, ifdesired. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, or in a liposomal preparation. Formulations for parenteraladministration may be formulated to be immediate and/or modifiedrelease. Modified release formulations include delayed-, sustained-,pulsed-, controlled-, targeted and programmed release.

For example, in one aspect, sterile injectable solutions can be preparedby incorporating the isolated antagonistic antigen binding protein inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, methods of preparation such as vacuum drying andfreeze-drying yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin. In various embodiments, the injectable compositions will beadministered using commercially available disposable injectable devices.

The isolated antagonistic antigen binding protein of the presentdisclosure can be administered intranasally or by inhalation, typicallyin the form of a dry powder (either alone, as a mixture, or as a mixedcomponent particle, for example, mixed with a suitable pharmaceuticallyacceptable carrier) from a dry powder inhaler, as an aerosol spray froma pressurized container, pump, spray, atomiser (preferably an atomiserusing electrohydrodynamics to produce a fine mist), or nebulizer, withor without the use of a suitable propellant, or as nasal drops.

The pressurized container, pump, spray, atomizer, or nebulizer generallycontains a solution or suspension of an isolated antagonistic antigenbinding protein of the disclosure comprising, for example, a suitableagent for dispersing, solubilizing, or extending release of the active,a propellant(s) as solvent.

Prior to use in a dry powder or suspension formulation, the drug productis generally micronized to a size suitable for delivery by inhalation(typically less than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenization, or spray drying.

Capsules, blisters and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the isolated antagonisticantigen binding protein of the disclosure, a suitable powder base and aperformance modifier.

Suitable flavours, such as menthol and levomenthol, or sweeteners, suchas saccharin or saccharin sodium, may be added to those formulations ofthe disclosure intended for inhaled/intranasal administration.

Formulations for inhaled/intranasal administration may be formulated tobe immediate and/or modified release. Modified release formulationsinclude delayed-, sustained-, pulsed-, controlled-, targeted andprogrammed release.

In the case of dry powder inhalers and aerosols, the dosage unit isdetermined by means of a valve which delivers a metered amount. Units inaccordance with the disclosure are typically arranged to administer ametered dose or “puff” of an antibody of the disclosure. The overalldaily dose will typically be administered in a single dose or, moreusually, as divided doses throughout the day.

The isolated antagonistic antigen binding protein of the presentdisclosure may also be formulated for an oral administration. Oraladministration may involve swallowing, so that the compound enters thegastrointestinal tract, and/or buccal, lingual, or sublingualadministration by which the compound enters the blood stream directlyfrom the mouth. Formulations suitable for oral administration includesolid, semi-solid and liquid systems such as tablets; soft or hardcapsules containing multi- or nano-particulates, liquids, or powders;lozenges (including liquid-filled); chews; gels; fast dispersing dosageforms; films; ovules; sprays; and buccal/mucoadhesive patches.

Pharmaceutical compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents inorder to provide a pharmaceutically elegant and palatable preparation.For example, to prepare orally deliverable tablets, the isolatedantagonistic antigen binding protein is mixed with at least onepharmaceutical carrier, and the solid formulation is compressed to forma tablet according to known methods, for delivery to thegastrointestinal tract. The tablet composition is typically formulatedwith additives, e.g. a saccharide or cellulose carrier, a binder such asstarch paste or methyl cellulose, a filler, a disintegrator, or otheradditives typically usually used in the manufacture of medicalpreparations. To prepare orally deliverable capsules, DHEA is mixed withat least one pharmaceutical carrier, and the solid formulation is placedin a capsular container suitable for delivery to the gastrointestinaltract. Compositions comprising isolated antagonistic antigen bindingprotein may be prepared as described generally in Remington'sPharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa.18042) at Chapter 89, which is herein incorporated by reference.

In various embodiments, the pharmaceutical compositions are formulatedas orally deliverable tablets containing isolated antagonistic antigenbinding protein in admixture with non-toxic pharmaceutically acceptablecarriers which are suitable for manufacture of tablets. These carriersmay be inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, maize starch, gelatin or acacia, andlubricating agents, for example, magnesium stearate, stearic acid, ortalc. The tablets may be uncoated or they may be coated with knowntechniques to delay disintegration and absorption in thegastrointestinal track and thereby provide a sustained action over alonger period of time. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

In various embodiments, the pharmaceutical compositions are formulatedas hard gelatin capsules wherein the isolated antagonistic antigenbinding protein is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate, or kaolin or as soft gelatincapsules wherein the isolated antagonistic antigen binding protein ismixed with an aqueous or an oil medium, for example, arachis oil, peanutoil, liquid paraffin or olive oil.

Liquid formulations include suspensions, solutions, syrups and elixirs.Such formulations may be employed as fillers in soft or hard capsules(made, for example, from gelatin or hydroxypropylmethylcellulose) andtypically comprise a carrier, for example, water, ethanol, polyethyleneglycol, propylene glycol, methylcellulose, or a suitable oil, and one ormore emulsifying agents and/or suspending agents. Liquid formulationsmay also be prepared by the reconstitution of a solid, for example, froma sachet.

Any method for administering peptides, proteins or antibodies acceptedin the art may suitably be employed for administering the isolatedantagonistic antigen binding protein of the disclosure.

Methods of Treatment

Due to their interaction with the glucagon receptor, the present antigenbinding and antagonizing proteins are useful for lowering blood glucoselevels by regulating gluconeogenesis and glycogenlysis and also for thetreatment of a wide range of conditions and disorders in which blockingthe interaction of glucagon with its receptor is beneficial, while alsoreducing and or eliminating one or more of the unwanted side effectsassociated with the current treatments.

An antagonistic antigen binding protein, in particular a human antibodyaccording to the present disclosure, need not effect a complete cure, oreradicate every symptom or manifestation of a disease, to constitute aviable therapeutic agent. As is recognized in the pertinent field, drugsemployed as therapeutic agents may reduce the severity of a givendisease state, but need not abolish every manifestation of the diseaseto be regarded as useful therapeutic agents. Similarly, aprophylactically administered treatment need not be completely effectivein preventing the onset of a condition in order to constitute a viableprophylactic agent. Simply reducing the impact of a disease (forexample, by reducing the number or severity of its symptoms, or byincreasing the effectiveness of another treatment, or by producinganother beneficial effect), or reducing the likelihood that the diseasewill occur or worsen in a subject, is sufficient. One embodiment of thedisclosure is directed to a method comprising administering to a subjectan isolated antagonistic antigen binding protein such as a humanantibody in an amount and for a time sufficient to induce a sustainedimprovement over baseline of an indicator that reflects the severity ofthe particular disorder.

Cardiovascular Diseases/Diabetic Cardiomyopathy/Heart Failure

Cardiovascular disease is a general name for a wide variety of diseases,disorders and conditions that affect the heart and/or blood vessels.Types of cardiovascular disease includes angina, heart attack(myocardial infarction), atherosclerosis, heart failure, cardiovasculardisease, rheumatic heart disease, cardiac arrhythmias (abnormal heartrhythms), cerebrovascular disease, congenital heart defects,cardiomyopathy, left ventricular hypertrophy, right ventricularhypertrophy, post-infarction heart rupture, infections of the heart,coronary artery disease, peripheral arterial disease, renal arterystenosis, aortic aneurysm, myocardial diseases, heart valve disorders,myocarditis, and pericarditis.

Heart failure is a clinical syndrome characterized by the failure of theheart to pump sufficient blood to meet the body's systemic demands. Theheart contracts and relaxes with each heartbeat—these phases arereferred to as systole (the contraction phase) and diastole (therelaxation phase). Systolic heart failure (SHF) is characterized by lowejection fraction. In patients with diastolic heart failure (DHF),contraction may be normal but relaxation of the heart may be impaired.This impairment is generally caused by a stiffening of the ventricles.Such impairment is referred to as diastolic dysfunction and if severeenough to cause pulmonary congestion (increased pressure and fluid inthe blood vessels of the lungs), diastolic heart failure. DHF patientsdiffer from those patients with SHF, in that DHF patients may have a“normal” ejection fraction. However, because the ventricle doesn't relaxnormally, the pressure within the ventricle increases and the bloodfilling the ventricle exceeds what is “normal”. People with certaintypes of cardiomyopathy may also have diastolic dysfunction. Leftventricular hypertrophy refers to a thickening of the left ventricle asa result of increased left ventricular load. Left ventricularhypertrophy can be a significant marker for cardiovascular disorders andmost common complications include arrhythmias, heart failure, ischemicheart disease, and sudden death. Although left ventricular hypertrophy(LVH) increases naturally with age, it is more common in people who havehigh blood pressure or have other heart problems. Because LVH usuallydevelops in response to hypertension, current treatment and preventionmainly includes managing hypertension. Typical diagnosis involves theuse of echocardiograms (ECHO) and electrocardiograms (ECG).

In one aspect, the present disclosure comprises a method for treating orpreventing heart failure in a subject, comprising administering to thesubject a therapeutically effective amount of an isolated antagonisticantigen binding protein that specifically binds to the human glucagonreceptor. In various embodiments, an isolated antagonistic antigenbinding protein of the present disclosure is a fully human anti-GCGRantibody that comprises a heavy chain sequence as set forth in SEQ IDNO: 51 and a light chain as set forth in SEQ ID NO: 52.

In one aspect, the present disclosure comprises a method for treatingpost-myocardial infarction heart failure in a subject in need thereofcomprising administering to the subject a therapeutically effectiveamount of an isolated antagonistic antigen binding protein thatspecifically binds to the human glucagon receptor. In variousembodiments, an isolated antagonistic antigen binding protein of thepresent disclosure is a fully human anti-GCGR antibody that comprises aheavy chain sequence as set forth in SEQ ID NO: 51 and a light chain asset forth in SEQ ID NO: 52.

In one aspect, the present discloses comprises a method of treatinglateral ventricular (LV) remodeling in a subject, comprisingadministering to a subject diagnosed with heart failure, or a subject atrisk of contracting heart failure, a therapeutically effective amount ofan isolated antagonistic antigen binding protein that specifically bindsto the human glucagon receptor. In various embodiments, an isolatedantagonistic antigen binding protein of the present disclosure is afully human anti-GCGR antibody that comprises a heavy chain sequence asset forth in SEQ ID NO: 51 and a light chain as set forth in SEQ ID NO:52.

Diabetic cardiomyopathy describes diabetes-associated changes in thestructure and function of the myocardium that is not directlyattributable to other confounding factors such as coronary arterydisease (CAD) or hypertension. It has been reported that in manypatients with type 2 diabetes, diabetes associated changes are amplifiedby the existence of these comorbidities, which likely will augment thedevelopment of left ventricular hypertrophy, increase the susceptibilityof the heart to ischemic injury and increase the overall likelihood ofdeveloping heart failure (Boudina and Abel, Rev Endocr Metab Disord.,11(1): 31-39, 2010 March). Several mechanisms have been implicated inthe pathogenesis of diabetic cardiomyopathy, and the mechanismsresponsible for diabetic cardiomyopathy continue to be elucidated.Changes in myocardial structure, calcium signaling and metabolism areearly defects that have been described mainly in animal models and mayprecede clinically manifest cardiac dysfunction (Id). Diabeticcardiomyopathy in humans is characterized by diastolic dysfunction (DD),which may precede the development of systolic dysfunction (SD).

Diabetic nephropathies are nerve damaging disorders that are a commonserious complication of diabetes mellitus. There are four main types ofdiabetic neuropathy: peripheral, autonomic, radiculoplexus, andmononeuropathy. Individuals may have just one type or symptoms ofseveral types. Most symptoms develop gradually, and problems may not benoticed until considerable damage has occurred. Prolonged exposure tohigh blood sugar can damage delicate nerve fibers, causing diabeticneuropathy. Accordingly, it may to possible to prevent diabeticneuropathy or slow its progress with tight blood sugar control.

In various embodiments, the present disclosure comprises a method fortreating or preventing heart failure and associated conditions in asubject, comprising administering to a subject diagnosed with diabetesmellitus or a subject at risk of developing diabetes mellitus, atherapeutically effective amount of an isolated antagonistic antigenbinding protein that specifically binds to the human glucagon receptor.In various embodiments, the isolated antagonistic antigen bindingprotein comprises an antibody which comprises the amino acid sequenceencoding the heavy chain of SEQ ID NO: 51 and the amino acid sequenceencoding the light chain of SEQ ID NO: 52.

In one aspect, the present disclosure comprises a method for treatingdiabetic cardiomyopathy in a subject in need thereof comprisingadministering to the subject a therapeutically effective amount of anisolated antagonistic antigen binding protein that specifically binds tothe human glucagon receptor. In various embodiments, an isolatedantagonistic antigen binding protein of the present disclosure is afully human anti-GCGR antibody that comprises a heavy chain sequence asset forth in SEQ ID NO: 51 and a light chain as set forth in SEQ ID NO:52.

An isolated antagonistic antigen binding protein that specifically bindsthe human glucagon receptor, in particular, the fully human antibodiesof the disclosure, may be administered, e.g., once or more than once, atregular intervals over a period of time. In various embodiments, a fullyhuman antibody is administered over a period of at least once a month ormore, e.g., for one, two, or three months or even indefinitely. Fortreating chronic conditions, long-term treatment is generally mosteffective. However, for treating acute conditions, administration forshorter periods, e.g. from one to six weeks, may be sufficient. Ingeneral, the fully human antibody is administered until the subjectmanifests a medically relevant degree of improvement over baseline forthe chosen indicator or indicators.

One example of therapeutic regimens provided herein comprisesubcutaneous injection of an isolated antagonistic antigen bindingprotein once a week, or once every two weeks, at an appropriate dosage,to treat a condition in which blood glucose levels play a role. Weeklyor monthly administration of isolated antagonistic antigen bindingprotein would be continued until a desired result is achieved, e.g., thesubject's symptoms subside. Treatment may resume as needed, or,alternatively, maintenance doses may be administered.

A subject's levels of blood glucose may be monitored before, duringand/or after treatment with an isolated antagonistic antigen bindingprotein such as a human antibody, to detect changes, if any, in theirlevels. For some disorders, the incidence of elevated blood glucose mayvary according to such factors as the stage of the disease. Knowntechniques may be employed for measuring glucose levels. Glucagon levelsmay also be measured in the subject's blood using known techniques, forexample, ELISA.

A therapeutically effective dose can be estimated initially from cellculture assays by determining an IC₅₀. A dose can then be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC₅₀ as determined in cell culture. Such information can beused to more accurately determine useful doses in humans. Levels inplasma may be measured by, e.g., HPLC or immunoassays using theanti-idiotypic antibodies specific to the therapeutic drug. The exactcomposition, route of administration and dosage can be chosen by theindividual physician in view of the subject's condition.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses (multiple or repeat ormaintenance) can be administered over time and the dose can beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

The specification for the dosage unit forms of the present disclosurewill be dictated primarily by the unique characteristics of the antibodyand the particular therapeutic or prophylactic effect to be achieved.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a subject may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the subject.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a subject in practicingthe present disclosure.

It is to be noted that dosage values may vary with the type and severityof the condition to be ameliorated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Further, the dosage regimen with the compositions of this disclosure maybe based on a variety of factors, including the type of disease, theage, weight, sex, medical condition of the subject, the severity of thecondition, the route of administration, and the particular antibodyemployed. Thus, the dosage regimen can vary widely, but can bedetermined routinely using standard methods. For example, doses may beadjusted based on pharmacokinetic or pharmacodynamic parameters, whichmay include clinical effects such as toxic effects and/or laboratoryvalues. Thus, the present disclosure encompasses intra-subjectdose-escalation as determined by the skilled artisan. Determiningappropriate dosages and regimens are well-known in the relevant art andwould be understood to be encompassed by the skilled artisan onceprovided the teachings disclosed herein.

For administration to human subjects, the total monthly dose of theisolated antagonistic antigen binding protein of the disclosure can bein the range of 0.5-1200 mg per subject, 0.5-1100 mg per subject,0.5-1000 mg per subject, 0.5-900 mg per subject, 0.5-800 mg per subject,0.5-700 mg per subject, 0.5-600 mg per subject, 0.5-500 mg per subject,0.5-400 mg per subject, 0.5-300 mg per subject, 0.5-200 mg per subject,0.5-100 mg per subject, 0.5-50 mg per subject, 1-1200 mg per subject,1-1100 mg per subject, 1-1000 mg per subject, 1-900 mg per subject,1-800 mg per subject, 1-700 mg per subject, 1-600 mg per subject, 1-500mg per subject, 1-400 mg per subject, 1-300 mg per subject, 1-200 mg persubject, 1-100 mg per subject, or 1-50 mg per subject depending, ofcourse, on the mode of administration. For example, an intravenousmonthly dose can require about 1-1000 mg/subject. In certainembodiments, the isolated antagonistic antigen binding protein of thedisclosure can be administered at about 1-200 mg per subject, 1-150 mgper subject or 1-100 mg per subject. The total monthly dose can beadministered in single or divided doses and can, at the physician'sdiscretion, fall outside of the typical ranges given herein.

An exemplary, non-limiting monthly dosing range for a therapeutically orprophylactically effective amount of an isolated antagonistic antigenbinding protein of the disclosure can be 0.001 to 10 mg/kg, 0.001 to 9mg/kg, 0.001 to 8 mg/kg, 0.001 to 7 mg/kg, 0.001 to 6 mg/kg, 0.001 to 5mg/kg, 0.001 to 4 mg/kg, 0.001 to 3 mg/kg, 0.001 to 2 mg/kg, 0.001 to 1mg/kg, 0.010 to 10 mg/kg, 0.010 to 9 mg/kg, 0.010 to 8 mg/kg, 0.010 to 7mg/kg, 0.010 to 6 mg/kg, 0.010 to 5 mg/kg, 0.010 to 4 mg/kg, 0.010 to 3mg/kg, 0.010 to 2 mg/kg, 0.010 to 1 mg/kg, 0.1 to 10 mg/kg, 0.1 to 9mg/kg, 0.1 to 8 mg/kg, 0.1 to 7 mg/kg, 0.1 to 6 mg/kg, 0.1 to 5 mg/kg,0.1 to 4 mg/kg, 0.1 to 3 mg/kg, 0.1 to 2 mg/kg, 0.1 to 1 mg/kg, 1 to 10mg/kg, 1 to 9 mg/kg, 1 to 8 mg/kg, 1 to 7 mg/kg, 1 to 6 mg/kg, 1 to 5mg/kg, 1 to 4 mg/kg, 1 to 3 mg/kg, 1 to 2 mg/kg, or 1 to 1 mg/kg bodyweight. It is to be noted that dosage values may vary with the type andseverity of the condition to be ameliorated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

In various embodiments, the total dose administered will achieve aplasma antibody concentration in the range of, e.g., about 1 to 1000μg/ml, about 1 to 750 μg/ml, about 1 to 500 μg/ml, about 1 to 250 μg/ml,about 10 to 1000 μg/ml, about 10 to 750 μg/ml, about 10 to 500 μg/ml,about 10 to 250 μg/ml, about 20 to 1000 μg/ml, about 20 to 750 μg/ml,about 20 to 500 μg/ml, about 20 to 250 μg/ml, about 30 to 1000 μg/ml,about 30 to 750 μg/ml, about 30 to 500 μg/ml, about 30 to 250 μg/ml.

Toxicity and therapeutic index of the pharmaceutical compositions of thedisclosure can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effective dose is the therapeutic indexand it can be expressed as the ratio LD₅₀/ED₅₀. Compositions thatexhibit large therapeutic indices are generally preferred.

In various embodiments, single or multiple administrations of thepharmaceutical compositions are administered depending on the dosage andfrequency as required and tolerated by the subject. In any event, thecomposition should provide a sufficient quantity of at least one of theisolated antagonistic antigen binding protein disclosed herein toeffectively treat the subject. The dosage can be administered once butmay be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy.

The dosing frequency of the administration of the isolated antagonisticantigen binding protein pharmaceutical composition depends on the natureof the therapy and the particular disease being treated. The subject canbe treated at regular intervals, such as weekly or monthly, until adesired therapeutic result is achieved. Exemplary dosing frequenciesinclude, but are not limited to: once weekly without break; once weekly,every other week; once every 2 weeks; once every 3 weeks; weakly withoutbreak for 2 weeks, then monthly; weakly without break for 3 weeks, thenmonthly; monthly; once every other month; once every three months; onceevery four months; once every five months; or once every six months, oryearly.

Combination Therapy

As used herein, the terms “co-administration”, “co-administered” and “incombination with”, referring to the isolated antagonistic antigenbinding protein of the present disclosure and one or more othertherapeutic agent(s), is intended to mean, and does refer to and includethe following: simultaneous administration of such combination ofisolated antagonistic antigen binding protein of the disclosure andtherapeutic agent(s) to a subject in need of treatment, when suchcomponents are formulated together into a single dosage form whichreleases said components at substantially the same time to said subject;substantially simultaneous administration of such combination ofisolated antagonistic antigen binding protein of the disclosure andtherapeutic agent(s) to a subject in need of treatment, when suchcomponents are formulated apart from each other into separate dosageforms which are taken at substantially the same time by said subject,whereupon said components are released at substantially the same time tosaid subject; sequential administration of such combination of isolatedantagonistic antigen binding protein of the disclosure and therapeuticagent(s) to a subject in need of treatment, when such components areformulated apart from each other into separate dosage forms which aretaken at consecutive times by said subject with a significant timeinterval between each administration, whereupon said components arereleased at substantially different times to said subject; andsequential administration of such combination of isolated antagonisticantigen binding protein of the disclosure and therapeutic agent(s) to asubject in need of treatment, when such components are formulatedtogether into a single dosage form which releases said components in acontrolled manner whereupon they are concurrently, consecutively, and/oroverlappingly released at the same and/or different times to saidsubject, where each part may be administered by either the same or adifferent route.

Suitable pharmaceutical agents that may be used in combination with thecompounds of the present invention include antihypertensive agents andagents for treating chronic heart failure, atherosclerosis or relateddiseases. Such agents contemplated for use include, but are not limitedto, bimoclomol, angiotensin-converting enzyme inhibitors (such ascaptopril, enalapril, fosinopril, lisinopril, perindopril, quinapril,ramipril and the like), neutral endopeptidase inhibitors (such asthiorphan, omapatrilat, MDL-100240, fasidotril, sampatrilat, GW-660511,mixanpril, SA-7060, E-4030, SLV-306, ecadotril and the like),angiotensin receptor antagonists (such as candesartan cilexetil,eprosartan, irbesartan, losartan, olmesartan medoxomil, telmisartan,valsartan, tasosartan, enoltasosartan and the like),endothelin-converting enzyme inhibitors (such as CGS 35066, CGS 26303,CGS-31447, SM-19712 and the like), endothelin receptor antagonists (suchas tracleer, sitaxsentan, ambrisentan, L-749805, TBC-3214, BMS-182874,BQ-610, TA-0201, SB-215355, PD-180988, BMS-193884, darusentan, TBC-3711,bosentan, tezosentan, J-104132, YM-598, S-0139, SB-234551, RPR-118031A,ATZ-1993, RO-61-1790, ABT-546, enlasentan, BMS-207940 and the like),diuretic agents (such as hydrochlorothiazide, bendroflumethiazide,trichlormethiazide, indapamide, metolazone, furosemide, bumetamide,torsemide, chlorthalidone, metolazone, cyclopenthiazide,hydroflumethiazide, tripamide, mefruside, benzylhydrochlorothiazide,penflutizide, methyclothiazide, azosemide, etacrynic acid, torasemide,piretamide, meticrane, potassium canrenoate, spironolactone,triamterene, aminophylline, cicletanine, LLU-alpha, PNU-80873A,isosorbide, D-mannitol, D-sorbitol, fructose, glycerin, acetazolamide,methazolamide, FR-179544, OPC-31260, lixivaptan, conivaptan and thelike), calcium channel antagonists (such as amlodipine, bepridil,diltiazem, felodipine, isradipine, nicardipen, nimodipine, verapamil,S-verapamil, aranidipine, efonidipine, barnidipine, benidipine,manidipine, cilnidipine, nisoldipine, nitrendipine, nifedipine,nilvadipine, felodipine, pranidipine, lercanidipine, isradipine,elgodipine, azelnidipine, lacidipine, vatanidipine, lemildipine,diltiazem, clentiazem, fasudil, bepridil, gallopamil and the like),vasodilating antihypertensive agents (such as indapamide, todralazine,hydralazine, cadralazine, budralazine and the like), beta blockers (suchas acebutolol, bisoprolol, esmolol, propanolol, atenolol, labetalol,carvedilol, metoprolol and the like), sympathetic blocking agents (suchas amosulalol, terazosin, bunazosin, prazosin, doxazosin, propranolol,atenolol, metoprolol, carvedilol, nipradilol, celiprolol, nebivolol,betaxolol, pindolol, tertatolol, bevantolol, timolol, carteolol,bisoprolol, bopindolol, nipradilol, penbutolol, acebutolol, tilisolol,nadolol, urapidil, indoramin and the like), alpha-2-adrenoceptoragonists (such as clonidine, methyldopa, CHF-1035, guanabenz acetate,guanfacine, moxonidine, lofexidine, talipexole and the like), centrallyacting antihypertensive agents (such as reserpine and the like),thrombocyte aggregation inhibitors (such as warfarin, dicumarol,phenprocoumon, acenocoumarol, anisindione, phenindione, ximelagatran andthe like), antiplatelets agents (such as aspirin, clopidogrel,ticlopidine, dipyridamole, cilostazol, ethyl icosapentate, sarpogrelate,dilazep, trapidil, beraprost and the like), and neuregulins (NRG-1,NRG-2, NRG-3 and NRG-4) and isoforms thereof.

In various embodiments, the present disclosure comprises a method fortreating or preventing heart failure and associated conditions in asubject, comprising administering to a subject diagnosed with heartfailure, or a subject at risk of contracting heart failure, atherapeutically effective amount of an isolated antagonistic antigenbinding protein that specifically binds to the human glucagon receptor;and (b) a second agent composition. In various embodiments, an isolatedantagonistic antigen binding protein of the present disclosure is afully human anti-GCGR antibody that comprises a heavy chain sequence asset forth in SEQ ID NO: 51 and a light chain as set forth in SEQ ID NO:52, and the second agent composition is selected from a group consistingof: angiotensin-converting enzyme (ACE) inhibitors, β-adrenergicblocking agents, angiotension II receptor blockers (ARBs), diuretics,and digitalis. In various embodiments, an isolated antagonistic antigenbinding protein of the present disclosure is a fully human anti-GCGRantibody that comprises a heavy chain sequence as set forth in SEQ IDNO: 51 and a light chain as set forth in SEQ ID NO: 52, and the secondagent composition is selected from a group consisting of:angiotensin-converting enzyme (ACE) inhibitors, β-adrenergic blockingagents, angiotension II receptor blockers (ARBs), diuretics, anddigitalis. In various embodiments, an isolated antagonistic antigenbinding protein of the present disclosure is a fully human anti-GCGRantibody that comprises a heavy chain sequence as set forth in SEQ IDNO: 51 and a light chain as set forth in SEQ ID NO: 52, and the secondagent composition is an angiotensin-converting enzyme (ACE) inhibitor.In various embodiments, an isolated antagonistic antigen binding proteinof the present disclosure is a fully human anti-GCGR antibody thatcomprises a heavy chain sequence as set forth in SEQ ID NO: 51 and alight chain as set forth in SEQ ID NO: 52, and the second agentcomposition is an β-adrenergic blocking agent. In various embodiments,an isolated antagonistic antigen binding protein of the presentdisclosure is a fully human anti-GCGR antibody that comprises a heavychain sequence as set forth in SEQ ID NO: 51 and a light chain as setforth in SEQ ID NO: 52, and the second agent composition is a diuretic.In various embodiments, an isolated antagonistic antigen binding proteinof the present disclosure is a fully human anti-GCGR antibody thatcomprises a heavy chain sequence as set forth in SEQ ID NO: 51 and alight chain as set forth in SEQ ID NO: 52, and the second agentcomposition is digitalis.

In various embodiments, the combination therapy comprises administeringthe isolated antagonistic antigen binding protein composition and thesecond agent composition simultaneously, either in the samepharmaceutical composition or in separate pharmaceutical compositions.In various embodiments, isolated antagonistic antigen binding proteincomposition and the second agent composition are administeredsequentially, i.e., the isolated antagonistic antigen binding proteincomposition is administered either prior to or after the administrationof the second agent composition.

In various embodiments, the administrations of the isolated antagonisticantigen binding protein composition and the second agent composition areconcurrent, i.e., the administration period of the isolated antagonisticantigen binding protein composition and the second agent compositionoverlap with each other.

In various embodiments, the administrations of the isolated antagonisticantigen binding protein composition and the second agent composition arenon-concurrent. For example, in various embodiments, the administrationof the isolated antagonistic antigen binding protein composition isterminated before the second agent composition is administered. Invarious embodiments, the administration second agent composition isterminated before the isolated antagonistic antigen binding proteincomposition is administered.

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

Example 1

In this example, the present inventors evaluated the therapeuticpotential of a monoclonal antibody against the glucagon receptor on LVremodeling in post-myocardial infarction heart failure. In this example,the in vivo activity of an anti-GCGR antibody which comprises the heavychain sequence set forth in SEQ ID NO: 8 and the light chain sequenceset forth in SEQ ID NO: 9 (“REMD2.590”) is evaluated using myocardialinfarction induced C57BL/6 mice.

Myocardial infarction (MI) was induced in 54 C57BL6 male mice at the ageof 8-10 weeks old by occluding the left coronary artery. After MI, micewere randomly divided into three groups. Group 1 was treated with thevehicle PBS as a control (CON, n=18); Group 2 was treated with 7.5 mg/kgREMD2.59C mAb, s.c. at 2 hours and at 14 days post-MI (mAb, n=18); andGroup 3 was treated with glucagon (30 ng/kg body weight in 10% gelatin)three times per day for the first six days post MI (GLC, n=18). Shamanimals were used as a negative control.

Infarcted mice were checked for survival every day for 28 days. Survivalcurves were delineated over time. Fasting blood glucose measurements aretaken at 2 weeks post-MI. Cardiac function was monitored during thepost-MI recovery. At the end of the 28 day study, the mice wereeuthanized and the hearts were harvested for histological studies andventricular contraction (echo FS/EF), ventricular pressure andcontractility (catheter); ventricular remodeling, septum and LV wallthickness (echo), cardiac index (HW/BW), myocyte hypertrophy, fibrosis;infarct size, and apoptosis were determined.

Cardiac function was measured by echocardiography three times, the firstday after MI, then at 14 and 28 days post-MI. The first echo was used toscreen infarcted mice for no infarction. The mice with left ventricularfractional shortening greater than 40% were eliminated from the study.At the end of the experiment, ventricular function was measured byMicro-tip pressure transducer (catheter). For the histological studies,the heart was perfused with 10% formalin for fixation. Cardiac sectionsfrom the apex, middle, and base of the heart were used for Masson'strichrome staining (MTS) and TUNEL staining. Infarct size was measuredin MTS sections, and apoptosis was assessed in the sections after TUNELstaining. The protective effect of REMD2.59C mAb was assessed based oninfarct size and apoptosis. Fibrosis and myocyte size were measured inMTS sections for assessment of cardiac remodeling.

As depicted in FIG. 1, at the end of the study, MI caused 55% (10/18)mortality in CON mice, while REMD2.59C mAb treatment reduced themortality (11/18 or 61% of the mice survived), and glucagon treatmentincreased the mortality (6/18 or 33% of the mice survived). As depictedin FIG. 2, mAb significantly increased left ventricular fractionalshortening (FS) as compared to CON, while GLC reduced it as compared toCON. As depicted in FIG. 3, mAb attenuated left ventricular end systolicdiameter (LVESD) as compared to CON, while GLC increased it as comparedto CON. As depicted in FIG. 4, mAb attenuated left ventricular enddiastolic dimension (LVEDD) as compared to CON, while GLC increased itas compared to CON. As depicted in FIG. 5, mAb preserved leftventricular ischemic muscle as compared to CON, while GLC slightlyreduced it as compared to CON. As depicted in FIG. 6, mAb increased leftventricular contraction as compared to CON, while GLC decreased it ascompared to CON. As depicted in FIG. 7, mAb increased cardiaccontractility (left panel) and relaxation (right panel) as compared toCON, while GLC decreased both as compared to CON. As depicted in FIG. 8mAb attenuated cardiac remodeling as compared to CON while GLC slightlyincreased heart weight as compared to CON. As depicted in FIG. 9, mAbattenuated cardiac remodeling (decreased HW/BW) as compared to CON,while GLC slightly increased HW/BW as compared to CON. As depicted inFIG. 10, mAb decreased blood glucose levels in mice (fasted for 4 hours)as compared to CON at 2 weeks post-MI. As depicted in FIG. 11, apoptosiswas attenuated by mAb in the infarct area. As depicted in FIG. 12 andFIG. 13, fibrosis was attenuated by mAb in non-infarct area. As depictedin FIG. 14 and FIG. 15, monocyte hypertrophy was attenuated by mAb innon-infarct area. As depicted in FIG. 16, infarct size was decreased bymAb at weeks after MI. As depicted in FIG. 17, infarct area wasdecreased by mAb at weeks after MI.

Histology findings strongly support the protective effects of glucagonreceptor mAb against post-MI ventricular remodeling. Treatment withglucagon receptor mAb attenuated apoptosis in infarcted area anddecreased myocyte hypertrophy and fibrosis in non-infarct area. Infarctarea was decreased by glucagon receptor mAb at 4 weeks after MI,compared with PBS control. Cardiac remodeling was attenuated by themonoclonal antibody against glucagon receptor, which decreased HW/BW,increased LV contractility (+dp/dtm) and FS, and preserved LV wallthickness. Administering glucagon (the ligand) promoted cardiacremodeling and worsened heart function, suggesting that glucagonsignaling plays a role in post-MI remodeling.

The surprising results of these studies suggest that glucagon signalingis involved in post-MI ventricular remodeling, and that targetingglucagon receptor with an antagonistic mAb may be an effectivetherapeutic approach to prevention of heart failure after myocardialinfarction. The glucagon receptor antibody may be a powerful weapon infighting against adverse cardiac remodeling and heart failure after MI.

Example 2

In this example, the present inventors evaluated the potentialpreventive efficacy of a human anti-GCGR antibody on cardiomyopathyinduced by diabetes in db/db mice. In this example, the in vivo activityof a human anti-GCGR antibody which comprises the heavy chain sequenceset forth in SEQ ID NO: 51 and the light chain sequence set forth in SEQID NO: 52 (“REMD477”) was evaluated using C57BL/6/db/db male diabetesmice and db/+ male control mice.

In this 18 week study, 30 C57BL6/db/db diabetes male mice at the age of8-10 weeks and 18 db/+ male mice were randomly divided into four groups.In Group 1, db/+ mice (n=9) where treated with the vehicle PBS as acontrol (hereinafter “db/+ (PBS)”); in Group 2, db/+ mice (n=9) weretreated with REMD477 (hereinafter “db/+ (mAb)”); in Group 3, db/dbdiabetes mice (n=15) where treated with the vehicle PBS as a control(hereinafter “db/db (PBS)”); and in Group 4, db/db mice (n=15) weretreated with REMD477 (hereinafter “db/db (mAb)”). For Groups 2 and 4,REMD477 was dosed at 7 mg/kg weekly SC, and then 3 mg/kg weekly SC up to18 week, for a total of 13 injections. For Groups 1 and 3, 01. ml/mousePBS was dosed at the same frequency as treatment Groups 2 and 4. At theend of the 18 week study, the mice are euthanized and the hearts areharvested for histological studies.

Body weight, fasting blood glucose, and OGTT measurements are taken at0, 4, 8, 12 and 18 weeks after treatment. Blood insulin levels and bloodglucagon levels were measured at 0, 6, 12 and 18 weeks after treatment.Cardiac function was measured by echocardiography at day 1 aftertreatment, then at 6, 12 and 18 weeks post treatment. The first echo wasused to screen infarcted mice for no infarction. The mice with leftventricular fractional shortening greater than 40% were eliminated fromthe study. Cardiac function changes (e.g., ventricular systolic(echo/FS/EF) and diastolic (echo E/A and IRT) function, ventricularpressure and contractility (catheter), and cardiac output (echo)) aremonitored at 0, 6, 12 and 18 weeks after treatment. LV remodeling ismeasured by Micro-tip pressure transducer (catheter) at 18 weeks aftertreatment, e.g., left ventricular diastole pressure (mmHg) and cardiacindex (HW/BW) is measured at 18 weeks after treatment by Micro-tipcatheter. Serum level GLP-1 and GHbAc1 measurements are taken at day 0and at 18 weeks after treatment.

For the histological studies, the heart is perfused with 10% formalinfor fixation. Cardiac sections from the apex, middle, and base of theheart are used for Masson's trichrome staining (MTS) and TUNEL staining.Infarct size is measured in MTS sections, and apoptosis is assessed inthe sections after TUNEL staining. Fibrosis and myocyte size weremeasured in MTS sections for assessment of cardiac remodeling.

As depicted in FIG. 18, mAb has significant effects on lowering fastblood glucose levels, and is capable of returning blood concentrationsto normal as early as 3-4 weeks and capable of maintaining normal levelsfor 14 weeks, without insulin. As depicted in FIG. 19, the OGTT showedthat the glucose tolerance capacity has been significantly improved. Asdepicted in FIG. 20, mAb markedly attenuated left ventricular dilation(LVESD) as compared to PBS in the diabetic db/db mice, restored valuesto that of control db/+ mice and maintained the values for 18 weeks. Asdepicted in FIG. 21, mAb attenuated left ventricular end diastolicdimension (LVEDD) as compared to PBS at 18 weeks. As depicted in FIG.22, mAb significantly increased left ventricular fractional shortening(FS) as compared to PBS in the diabetic db/db, restored values to thatof control db/+ mice and maintained the values for 18 weeks. As depictedin FIG. 23, mAb markedly attenuated interventricular septal end diastole(IVSD) as compared to PBS in the diabetic db/db mice, restored values tothat of control db/+ mice and maintained the values for 18 weeks. Asdepicted in FIG. 24, mAb markedly reduced the ratios of Early (E) tolate (A) ventricular filling velocities (E/A) as compared to PBS in thediabetic db/db mice, restored values to that of control db/+ mice andmaintained the values for 18 weeks. As depicted in FIG. 25, mAb markedlyincreased Cardiac Output (CO, ml/min) as compared to PBS in the diabeticdb/db mice, restored values to that of control db/+ mice and maintainedthe values for 18 weeks. As depicted in FIG. 26, mAb markedly reducedIsovolumic Relaxation Time (IVRT, ms) as compared to PBS in the diabeticdb/db mice, restored values to that of control db/+ mice and maintainedthe values for 18 weeks. As depicted in FIG. 27, there was nostatistical significant difference on heart beat rate for mAb comparedto PBS control. As depicted in FIG. 28, mAb markedly increased leftventricular diastole pressure (LVDP, mmHg, measured by Mikro-tipcatheter) as compared to PBS in the diabetic db/db mice at week 18, andrestored values to nearly that of control db/+ mice. As depicted in FIG.29, +dp/dtm and −dp/dtm values were markedly improved by treatment ofmAb as compared to PBS at week 18 and mAb restored values to nearly thatof control db/+ mice. As depicted in FIG. 30, after treatment using mAb,the blood GLP-1 was significantly elevated compared to PBS treated db/dbmice, and the blood GHbA1c was significantly decreased compared to PBStreated db/db mice. As depicted in FIG. 31, mAb treatment resulted in asmall reduction in heart weight and body weight compared to PBS at week18. As depicted in FIG. 32, there was no significant differences betweentreatment groups in blood insulin levels at week 18.

Histology findings strongly support the potential preventive efficacy ofan anti-glucagon receptor mAb against cardiomyopathy induced bydiabetes. The surprising results of these studies suggest that glucagonsignaling is involved in cardiomyopathy induced by diabetes anddemonstrate that targeting glucagon receptor with an antagonistic mAbmay be an effective therapeutic approach to prevention of cardiomyopathyinduced by diabetes.

Example 3

In this example, the in vivo activity of an anti-GCGR antibody whichcomprises the heavy chain sequence set forth in SEQ ID NO: 8 and thelight chain sequence set forth in SEQ ID NO: 9 (“REMD2.59C”) isevaluated for efficacy in a miniature swine myocardial infarct model(Sinclair Research).

Miniature swine (young adult/female) are given a 7 day acclimationperiod and randomly divided into two groups. In Group 1, the swine aretreated with a single dose of vehicle PBS as a control (CON, n=5) and inGroup 2, the swine are treated with a single dose of 7.5 mg/kg REMD2.59CmAb (mAb, n=5). Prior to dose administration, each animal undergoessurgery (ligation of left ascending artery for 10-20 minutes) to createan occlusion and induce myocardial infarction. After 20 minutes, theocclusion is removed and the treatment administered during thereperfusion. The animals are maintained and handled in a stress-freeenvironment post-op.

Mortality/Morbidity observations are made twice daily. Variousassessments and parameters are made at baseline and prior to terminationor necropsy. These include body weight, clinical observations, clinicalpathology, clinical chemistry, hematology and coagulation parameters,and ECG/cardiac assessments. At the end of the study (28 days), limitednecropsy is performed and limited heart tissue collected forhistopathology (the middle of the infarct, the injected area and onemore at each extent of the infarcted area is evaluated) for histologicalstudies and ventricular contraction (echo FS/EF), ventricular pressureand contractility (catheter); ventricular remodeling, septum and LV wallthickness (echo), cardiac index (HW/BW), myocyte hypertrophy, fibrosis;infarct size, and apoptosis were determined.

Troponin level, size of ventricle, thickness of ventricle walls, andejection fraction by echocardiogram are the end points.

Additional Materials and Methods Preparation of Infarcted Mice

MI was induced in mice as described previously (Patten R D et al., Am JPhysiol., 1998; 274:H1812-1820. [PubMed: 9612394]). Briefly, the chestwas opened via a left thoracotomy. The left coronary artery wasidentified visually using a stereo microscope, and a 7-0 suture wasplaced around the artery 1-2 mm below the left auricle. Theelectrocardiogram (ECG) was monitored continuously. Permanent occlusionof the left coronary artery resulted from its ligation with the suture.Myocardial ischemia was confirmed by pallor in heart color andST-segment elevation. The chest was closed with 6-0 silk suture. Oncespontaneous respiration resumed, the endotracheal tube was removed. Theanimals were monitored until fully conscious. After they were returnedto their cages, standard chow and water were provided.

Echocardiography

In vivo cardiac function was assessed by transthoracic echocardiography(Acuson P300, 18 MHz transducer; Siemens) in conscious mice, asdescribed previously. The mouse heart was imaged in the two-dimensionalmode in the parasternal short axis at a papillary level. From this view,LV chamber dimensions were measured. Fractional shortening (FS) wascalculated from left ventricular (LV) end-diastolic diameter (EDD) andend-systolic diameter (ESD). M-mode EDD and ESD were averaged from threeto five beats. Studies and analyses were performed by investigatorsblinded to treatments.

Measurement of LV Pressure by Mikro-Tip Catheter

Mice were anesthetized with pentobarbital (40 mg/kg, ip). The chest wasopened via a left thoracotomy. The infarcted heart was exposed. A mouseMikro-tip catheter (SPR1000) was inserted into the left ventriclethrough non-infarcted ventricular wall. LV pressure was measured byPowerLab system (Adinstruments). Ventricular contractility wascalculated through Chart 7 software (Adinstruments).

Histological Studies

Hearts were fixed with 10% buffered formalin, embedded in paraffin, andsectioned at 6 μm. One middle longitudinal section per heart was stainedwith Masson's trichrome. Eight randomly selected fields (400×) from thenon-infarct area in the left ventricle were examined for fibrosis andmyocyte size under a microscope. Each group comprised 5-6 hearts, andminimal 40 fields were analyzed in each group by computerized planimetry(NIH Image J). To assess fibrosis, fibrotic blue area and wholemyocardial area were measured. The fibrotic area was presented as apercentage of fibrotic area to the myocardial area. Myocyte size wasmeasured in cross-sectioned muscle cells. Total 100-150 cells/heart wereanalyzed. Two methods were used to assess the size of the infarctedheart. Infarct area was calculated as a percentage of infarctedventricular area to total ventricular area using the front and backsides of the heart photos. Infarct size was measured as a percentage ofinfarcted ventricular wall length to total ventricular wall length usingcardiac sections. The observer was blinded to the origin of the cardiacsections.

TUNEL Assay

TUNEL assay was performed with the In Situ Apoptosis Detection Kit.Briefly, hearts were fixed by perfusion with 10% formalin solution,embedded in paraffin, and sectioned at 6 μm. One middle longitudinalsection per heart was taken for TUNEL staining. Proteinase K (20 μg/ml)was added to each slide. Endogenous peroxidases was inactivated bycovering sections with 2% hydrogen peroxide. After fixation, sectionswere incubated with TdT buffer at 37° C. for 30 min. Reactions wereterminated with 1×SSC. After being washed, slides were incubated withRTU streptavidin-HRP for 30 min. Positive signal was developed by addingDAB solution. After counterstained with RTU hematoxylin, slides werecovered by mounting medium and analyzed under a microscope. Each groupcomprised 5-6 hearts. Eight fields (400×) from the infarct area perheart were analyzed for positive cells and total cells usingcomputerized planimetry (NIH Image J). The degree of apoptosis waspresented as a percentage of positive cells to total cells.

All of the articles and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the articles and methods of this disclosure have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the articlesand methods without departing from the spirit and scope of thedisclosure. All such variations and equivalents apparent to thoseskilled in the art, whether now existing or later developed, are deemedto be within the spirit and scope of the disclosure as defined by theappended claims. All patents, patent applications, and publicationsmentioned in the specification are indicative of the levels of those ofordinary skill in the art to which the disclosure pertains. All patents,patent applications, and publications are herein incorporated byreference in their entirety for all purposes and to the same extent asif each individual publication was specifically and individuallyindicated to be incorporated by reference in its entirety for any andall purposes. The disclosure illustratively described herein suitablymay be practiced in the absence of any element(s) not specificallydisclosed herein. The terms and expressions which have been employed areused as terms of description and not of limitation, and there is nointention that in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the disclosure claimed. Thus, it should be understood thatalthough the present disclosure has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

SEQUENCE LISTINGS

The amino acid sequences listed in the accompanying sequence listing areshown using standard three letter code for amino acids, as defined in 37C.F.R. 1.822.

SEQ ID NO: 1 is the amino acid sequence of a human glucagon receptor(GCGR) molecule (Accession Number AA104855).

SEQ ID NO: 2 is the amino acid sequence encoding the heavy chainvariable region of a fully human anti-GCGR antibody. SEQ ID NO: 3 is theamino acid sequence encoding the light chain variable region of a fullyhuman anti-GCGR antibody.

SEQ ID NO: 4 is the amino acid sequence encoding the heavy chainvariable region of a fully human anti-GCGR antibody. SEQ ID NO: 5 is theamino acid sequence encoding the light chain variable region of a fullyhuman anti-GCGR antibody.

SEQ ID NO: 6 is the amino acid sequence encoding the heavy chainvariable region of a fully human anti-GCGR antibody. SEQ ID NO: 7 is theamino acid sequence encoding the light chain variable region of a fullyhuman anti-GCGR antibody.

SEQ ID NO: 8 is the amino acid sequence encoding the heavy chain of achimeric anti-GCGR antibody. SEQ ID NO: 9 is the amino acid sequenceencoding the light chain of a chimeric anti-GCGR antibody.

SEQ ID NOS: 10-28 are amino acid sequences encoding the heavy chainvariable regions of various fully human anti-GCGR antibodies.

SEQ ID NOS: 29-47 are amino acid sequences encoding the light chainvariable regions of various fully human anti-GCGR antibodies.

SEQ ID NO: 48 is the amino sequence encoding the kappa light chainconstant region. SEQ ID NO: 49 is the amino sequence encoding the lambdalight chain constant region.

SEQ ID NO: 50 is the amino sequence encoding the IgG2 heavy chainconstant region.

SEQ ID NO: 51 is the amino acid sequence encoding the heavy chain of ahuman anti-GCGR antibody. SEQ ID NO: 52 is the amino acid sequenceencoding the light chain of a human anti-GCGR antibody.

SEQUENCE LISTINGSSEQ ID NO: 1-Amino acid sequence of a human glucagon receptor (GCGR) moleculeMPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQPWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRSAKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPFSEQ ID NO: 2-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSSEQ ID NO: 3-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKSEQ ID NO: 4-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSSEQ ID NO: 5-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFVTYYCLQHNSNPLTFGGGTKVEIKSEQ ID NO: 6-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSSEQ ID NO: 7-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKSEQ ID NO: 8-Amino acid sequence of a heavy chain of a chimeric antibody thatbinds GCGR MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKSEQ ID NO: 9-Amino acid sequence of a light chain of a chimeric antibody thatbinds GCGRMDMRVPAQLLGLLLLWFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECSEQ ID NO: 10-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVILSDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYEILTGYGYYGMDVWGQGTTVTV SSSEQ ID NO: 11-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVILNDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYEILTGYGYYGMDVWGQGTTVTV SSSEQ ID NO: 12-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNGAAWNWIRQSPSRGLEWLGRTYYRSKWYYDYAGSVKSRININPDTSKNQFSLQVNSVTPEDTAVYYCTRDRSSGWNEGYYYYGMDVWGQG TTVTVSSSEQ ID NO: 13-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVRQAPGKGLEWVAVLSSDGNNKYCADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCAREEVYYDILTGYYDYYGMDVWGQGTTV TVSSSEQ ID NO: 14-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQESGPGLVKPSETLSLTCTVSGGSISTYFWTWIRQFPGKGLEWIGYIFYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGYYDILTGEDYSYGMDVWGQGTTVTVSSSEQ ID NO: 15-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQQSGPGLVKPSQILSLTCAISGDRVSSNGAAWNWIRQSPSRGLEWLGRTYYRSKWYYDYAGSVKSRININPDTSKNQFSLQVNSVTPEDTAVYYCARDRSSGWNEGYYYYGMDVWGQGT TVTVSSSEQ ID NO: 16-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLQESGPGLVKPSETLSLTCTVSGGSISTYFWTWIRQFPGEGLEWIGYIFYSGNTNYNPSLTSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGYYDILTGEDYSYGIDVWGQGTTVTVSSSEQ ID NO: 17-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFIFSSYGMHWVRQAPGKGLEWVAVISNDGSNKYYADFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDYDILTGNGVYGMDVWGQGTTVTV SSSEQ ID NO: 18-Amino acid sequence of a HCVR of a human antibody that binds GCGREVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMNWVRQAPGKGLEWVSYISGSSSLIYYADSVKGRFTISRDNAKNSLYLHMNSLRDEDTAVYYCARARYNWNDYYGMDVWGQGTTVTVSSSEQ ID NO: 19-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGIHWVRQAPGKGLEWVAGIWYDGSNKYYADSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARLFDAFDIWGQGTMVTVSSSEQ ID NO: 20-Amino acid sequence of a HCVR of a human antibody that binds GCGREVQLVESGGGLVQPGGSLRLSCAASGFIFSSYTMNWVRQAPGKGLEWVSYISSSSSLIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARSDYYGSGSYYKGNYYGMDVWGQGTTV TVSSSEQ ID NO: 21-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVTIIWSDGINKYYADSVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCARERGLYDILTGYYDYYGIDVWGQGTTVT VSSSEQ ID NO: 22-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVTIIWSDGINKYYADSVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCARERGLYDILTGYYDYYGIDVWGQGTTVT VSSSEQ ID NO: 23-Amino acid sequence of a HCVR of a human antibody that binds GCGREVQLVESGGGLVKPGGSLRLSCAASGITFRSYSMNWVRQAPGKGLEWVSAISSSSSYIYYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCARGRYGMDVWGQGTTVTVSSSEQ ID NO: 24-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGSTFRSYDMHWVRQAPGKGLEWVAVISYDGSNKYYGDSVKGRLTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQYDILTGYSSDAFDIWGQGTMVTV SSSEQ ID NO: 25-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSHKYYEDSVKGRFTISRDNSKNTLYLQMNSLRADDTGVYYCARVGYGSGWYEYYYHYGMDVWGQGT TVTVSSSEQ ID NO: 26-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTT VTVSSSEQ ID NO: 27-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTT VTVSSSEQ ID NO: 28-Amino acid sequence of a HCVR of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGITFSSYGMHWVRQAPGKGLEWVASIWYDGSNKYYVDSVKGRFTIFRDNSKKTLYLQMNRLRAEDTAVYYCARLGGGFDYWGQGTLVTVSSSEQ ID NO: 29-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQKKPGKAPKSLIYVVSSLQSGVPSRFSGSGSGTDFTLTINNLQPEDFATYYCQQYNHYPLTFGGGTRVEIKRSEQ ID NO: 30-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQRPGKAPKSLIYVVSSLQSGVPSRFSGSGSGTDFTLTISNLQPEDFATYFCQQYNHYPLTFGGGTKVEIKRSEQ ID NO: 31-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQFPSSLSASIGDRVTITCQASQDISNFLNWFQQKPGKAPKLLIYDASDLETGVPSRFSGSGAGTDFTFTISSLQPEDIATYFCQQYDDLPLTFGGGTRVDIKRSEQ ID NO: 32-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSNPLTFGGGTKVEIKRSEQ ID NO: 33-Amino acid sequence of a LCVR of a human antibody that binds GCGRQNVLTQSPGTLSLSPGERVTLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGVSSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGNSPFTFGPGTKVDIKRSEQ ID NO: 34-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQFPSSLSASIGDRVTITCQASQDISNFLNWFQQKPGKAPKLLIYDASDLETGVPSRFSGSGAGTDFTFTISSLQPEDVATYFCQQYDNLPLTFGGGTKVDIKRSEQ ID NO: 35-Amino acid sequence of a LCVR of a human antibody that binds GCGRENVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLIFGVSSRATGIPDRFSGSGSGTDFSLTISRLEPEDFAVYYCQQYGNSPFTFGPGTKVDIKRSEQ ID NO: 36-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIDMYLAWFQQKPGKAPKSLIYAASSLQSGVPSKFSGSGFGTDFTLTISSLQPEDFATYYCQQYNIFPFTFGPGTKVDVKRSEQ ID NO: 37-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRSEQ ID NO: 38-Amino acid sequence of a LCVR of a human antibody that binds GCGRKIVMTQTPLALPVIPGEPASISCRSSQSLVDSDDGDTYLDWYLQKPGQSPQVLIHRLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMHRIEFPFTFGGGTKVEIKRSEQ ID NO: 39-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQRPGKAPKRLIYAASSLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRSEQ ID NO: 40-Amino acid sequence of a LCVR of a human antibody that binds GCGRGIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMEALQTMCSFGQGTKLEIKRSEQ ID NO: 41-Amino acid sequence of a LCVR of a human antibody that binds GCGRGIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMEALQTMSSFGQGTKLEIKRSEQ ID NO: 42-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIVMTQTPLFLPVTPGEPASISCRSSQTLLDSDDGNTYLDWYLQKPGQSPQRLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQHIEFPSTFGQGTRLEIKRSEQ ID NO: 43-Amino acid sequence of a LCVR of a human antibody that binds GCGRSYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQSTKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVLGSEQ ID NO: 44-Amino acid sequence of a LCVR of a human antibody that binds GCGRNIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKNYLFWYLQKPGQSPQLLIYEVSYRFSGVPDRFSGSGSGTDFSLKISRVEAEDVGVYYCMQNIQPPLTFGQGTRLEIKRSEQ ID NO: 45-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRSEQ ID NO: 46-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRSEQ ID NO: 47-Amino acid sequence of a LCVR of a human antibody that binds GCGRDIVLTQTPLSLPVTPGEPASISCRSSQSLLDRDDGDTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFSLKISRVEAEDVGVYYCMQRIEFPFTFGPGTKVDIKRSEQ ID NO: 48-Amino acid sequence of the constant light chain kappa regionRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 49-Amino acid sequence of the constant light chain lambda regionGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSEQ ID NO: 50-Amino sequence of the IgG2 heavy chain constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 51-Amino acid sequence of a HC of a human antibody that binds GCGRQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 52-Amino acid sequence of a LC of a human antibody that binds GCGRDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

What is claimed is:
 1. A method for treating heart failure in a subjectin need thereof comprising administering to the subject atherapeutically effective amount of an isolated antagonistic antigenbinding protein that specifically binds to the human glucagon receptor.2. A method according to claim 1, wherein the heart failure to betreated is selected from the group consisting of post-myocardialinfarction heart failure and diabetic cardiomyopathy heart failure.
 3. Amethod according to claim 2, wherein the heart failure to be treated ispost-myocardial infarction heart failure.
 4. A method according to claim2, wherein the heart failure to be treated is diabetic cardiomyopathyheart failure.
 5. A method according to claim 1, wherein the isolatedantagonistic antigen binding protein comprises an isolated antagonisticantibody or antibody fragment selected from the group consisting of afully human antibody, a humanized antibody, a chimeric antibody, amonoclonal antibody, a polyclonal antibody, a recombinant antibody, anantigen-binding antibody fragment, a Fab, a Fab′, a Fab₂, a Fab′₂, aIgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody,a diabody, and a hemibody, and wherein the isolated antagonisticantibody or antibody fragment specifically binds to a human glucagonreceptor with a dissociation constant (K_(D)) of at least about 1×10⁻⁷M, at least about 1×10⁻⁸ M, at least about 1×10⁻⁸ M, at least about1×10⁻¹⁰ M, at least about 1×10⁻¹¹ M, or at least about 1×10⁻¹² M.
 6. Amethod according to claim 5, wherein the isolated antagonistic antibodyis a fully human antibody.
 7. A method according to claim 6, wherein thefully human antibody comprises a human anti-GCGR antibody whichcomprises the amino acid sequence encoding the heavy chain variableregion of SEQ ID NO: 2 and the amino acid sequence encoding the lightchain variable region of SEQ ID NO:
 3. 8. A method according to claim 6,wherein the fully human antibody comprises a human anti-GCGR antibodywhich comprises the amino acid sequence encoding the heavy chainvariable region of SEQ ID NO: 4 and the amino acid sequence encoding thelight chain variable region of SEQ ID NO:
 5. 9. A method according toclaim 6, wherein the fully human antibody comprises a human anti-GCGRantibody which comprises the amino acid sequence encoding the heavychain variable region of SEQ ID NO: 6 and the amino acid sequenceencoding the light chain variable region of SEQ ID NO:
 7. 10. A methodaccording to claim 6, wherein the fully human antibody comprises a heavychain variable region having the amino acid sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO:27, and SEQ ID NO:
 28. 11. A method according to claim 6, wherein thefully human antibody comprises a light chain variable region having theamino acid sequence selected from the group consisting of SEQ ID NO: 29,SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO:
 47. 12. A methodaccording to claim 6, wherein the fully human antibody comprises a humananti-GCGR antibody which comprises the amino acid sequence encoding theheavy chain variable region of SEQ ID NO: 28 and the amino acid sequenceencoding the light chain variable region of SEQ ID NO:
 47. 13. A methodaccording to claim 6, wherein the fully human antibody comprises a humananti-GCGR antibody which comprises the amino acid sequence encoding theheavy chain of SEQ ID NO: 51 and the amino acid sequence encoding thelight chain of SEQ ID NO:
 52. 14. A method according to claim 1, whereinthe therapeutically effective amount of the isolated antagonisticantigen binding protein is selected from: 0.001 to 100 mg/kg, 0.001 to90 mg/kg, 0.001 to 80 mg/kg, 0.001 to 70 mg/kg, 0.001 to 60 mg/kg, 0.001to 50 mg/kg, 0.001 to 40 mg/kg, 0.001 to 30 mg/kg, 0.001 to 20 mg/kg,0.001 to 10 mg/kg, 0.001 to 5 mg/kg, 0.001 to 4 mg/kg, 0.001 to 3 mg/kg,0.001 to 2 mg/kg, 0.001 to 1 mg/kg, 0.010 to 50 mg/kg, 0.010 to 40mg/kg, 0.010 to 30 mg/kg, 0.010 to 20 mg/kg, 0.010 to 10 mg/kg, 0.010 to5 mg/kg, 0.010 to 4 mg/kg, 0.010 to 3 mg/kg, 0.010 to 2 mg/kg, 0.010 to1 mg/kg, 0.1 to 50 mg/kg, 0.1 to 40 mg/kg, 0.1 to 30 mg/kg, 0.1 to 20mg/kg, 0.1 to 10 mg/kg, 0.1 to 5 mg/kg, 0.1 to 4 mg/kg, 0.1 to 3 mg/kg,0.1 to 2 mg/kg, 0.1 to 1 mg/kg, 0.5 to 50 mg/kg, 0.5 to 40 mg/kg, 0.5 to30 mg/kg, 0.5 to 20 mg/kg, 0.5 to 10 mg/kg, 0.5 to 5 mg/kg, 0.5 to 4mg/kg, 0.5 to 3 mg/kg, 0.5 to 2 mg/kg, 0.5 to 1 mg/kg, 1 to 50 mg/kg, 1to 40 mg/kg, 1 to 30 mg/kg, 1 to 20 mg/kg, 1 to 10 mg/kg, 1 to 5 mg/kg,1 to 4 mg/kg, 1 to 3 mg/kg, 1 to 2 mg/kg, and 1 mg/kg body weight perweek.
 15. A method according to claim 14, wherein the therapeuticallyeffective amount of the isolated antagonistic antigen binding protein is0.01 to 10 mg/kg body weight per week.
 16. A method according to claim1, said method further comprising administering an anti-heart failuredrug to the subject, wherein the ant-heart failure drug is selected froma group consisting of: angiotensin-converting enzyme (ACE) inhibitors,β-adrenergic blocking agents, angiotension II receptor blockers (ARBs),diuretics, and digitalis.
 17. A method of treating lateral ventricular(LV) remodeling comprising administration of a therapeutically effectiveamount of an isolated antagonistic antigen binding protein thatspecifically binds to the human glucagon receptor.
 18. A methodaccording to claim 17, said method further comprising administering ananti-heart failure drug to the subject, wherein the ant-heart failuredrug is selected from a group consisting of: angiotensin-convertingenzyme (ACE) inhibitors, β-adrenergic blocking agents, angiotension IIreceptor blockers (ARBs), diuretics, and digitalis.
 19. A methodaccording to claim 1, wherein the isolated antagonistic antigen bindingprotein is admixed with a pharmaceutically acceptable carrier to form apharmaceutical composition for systemic administration to the subject.20. A method according to claim 19, wherein the systemic administrationis selected from: intravenous injection, intramuscular injection,subcutaneous injection, intraperitoneal injection, transdermalinjection, intraarterial injection, intrasternal injection, intrathecalinjection, intraventricular injection, intraurethral injection,intracranial injection, intrasynovial injection or via infusions.